Engineered immunoglobulin heavy chain-light chain pairs and uses thereof

ABSTRACT

The present invention provides heterodimer pairs that can comprise a first heterodimer and a second heterodimer wherein each heterodimer comprises an immunoglobulin heavy chain or fragment thereof and an immunoglobulin light chain or fragment thereof. At least one of the heterodimers can comprise one or more amino acid modifications in the CH1 and/or CL domains, one or more amino acid modifications in the VH and/or VL domains, or a combination thereof. The modified amino acid(s) can be part of the interface between the light chain and heavy chain and are typically modified to create preferential pairing between each heavy chain and a desired light chain such that when the two heavy chains and two light chains of the heterodimer pair are co-expressed in a cell, the heavy chain of the first heterodimer preferentially pairs with one of the light chains rather than the other. Likewise, the heavy chain of the second heterodimer typically preferentially pairs with the second light chain rather than first.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is the divisional of U.S. patent application Ser. No.16/122,417, filed, Sep. 5, 2018, which is a divisional of Ser. No.14/648,222, filed May 28, 2015, now U.S. Pat. No. 10,077,298, which isthe U.S. National Entry of PCT/CA2013/050914, filed Nov. 28, 2013, whichclaims the benefit of U.S. Provisional Application No. 61/730,906, filedNov. 28, 2012, U.S. Provisional Application No. 61/761,641, filed Feb.6, 2013, U.S. Provisional Application No. 61/818,874, filed May 2, 2013,and U.S. Provisional Application No. 61/869,200, filed Aug. 23, 2013,the entire disclosure of each of which is hereby incorporated byreference in its entirety for all purposes.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Jan. 30, 2014, is named24897PCT_CRF_sequencelisting.txt, and is 91,025 bytes in size.

BACKGROUND

Bi-specific antibodies are capable of binding to two different epitopes.The epitopes can be on the same antigen, or each epitope can be on adifferent antigen. This feature of bi-specific antibodies makes them anattractive tool for various therapeutic applications where there is atherapeutic benefit to targeting or recruiting more than one molecule inthe treatment of disease. One of the approaches to form bi-specificantibody would involve concomitant expression of two unique antibodyheavy chains and two unique antibody light chains. Correctly formingbi-specific antibodies in a format that is similar to wild-type remainsa challenge, since antibody heavy chains have evolved to bind antibodylight chains in a relatively promiscuous manner. As a result of thispromiscuous pairing, concomitant expression of two antibody heavy chainsand two antibody light chains naturally leads to a scrambling of heavychain-light chain pairings. This mispairing remains a major challengefor the generation of bi-specific therapeutics, where homogeneouspairing is an essential requirement for good manufacturability andbiological efficacy.

Several approaches have been described to prepare bi-specific antibodiesin which specific antibody light chains or fragment pair with specificantibody heavy chains or fragments. A review of various approaches toaddress this problem can be found in Klein et al., (2012) mAbs 4:6,1-11. International Patent Application No. PCT/EP2011/056388 (WO2011/131746) describes an in vitro method for generating a heterodimericprotein in which asymmetrical mutations are introduced into the CH3regions of two monospecific starting proteins in order to drivedirectional “Fab-arm” or “half-molecule” exchange between twomonospecific IgG4- or IgG4-like antibodies upon incubation underreducing conditions.

Schaefer et al. (Roche Diagnostics GmbH), describe a method to assembletwo heavy and two light chains, derived from two existing antibodies,into human bivalent bi-specific IgG antibodies without use of artificiallinkers (PNAS (2011) 108(27): 11187-11192). The method involvesexchanging heavy chain and light chain domains within theantigen-binding fragment (Fab) of one half of the bi-specific antibody.

Strop et al. (Rinat-Pfizer Inc.), describe a method of producing stablebi-specific antibodies by expressing and purifying two antibodies ofinterest separately, and then mixing them together under specified redoxconditions (J. Mol. Biol. (2012) 420:204-19).

Zhu et al. (Genentech) have engineered mutations in the VL/VH interfaceof a diabody construct consisting of variant domain antibody fragmentscompletely devoid of constant domains, and generated a heterodimericdiabody (Protein Science (1997) 6:781-788). Similarly, Igawa et al.(Chugai) have also engineered mutations in the VL/VH interface of asingle-chain diabody to promote selective expression and inhibitconformational isomerization of the diabody (Protein Engineering, Design& Selection (2010) 23:667-677).

US Patent Publication No. 2009/0182127 (Novo Nordisk, Inc.) describesthe generation of bi-specific antibodies by modifying amino acidresidues at the Fc interface and at the CH1:CL interface of light-heavychain pairs that reduce the ability of the light chain of one pair tointeract with the heavy chain of the other pair.

SUMMARY

Described herein is an isolated antigen binding polypeptide constructcomprising at least a first heterodimer and a second heterodimer, thefirst heterodimer comprising a first immunoglobulin heavy chainpolypeptide sequence (H1), and a first immunoglobulin light chainpolypeptide sequence (L1); and the second heterodimer comprising asecond immunoglobulin heavy chain polypeptide sequence (H2), and asecond immunoglobulin light chain polypeptide sequence (L2), wherein atleast one of the H1 or L1 sequences of the first heterodimer is distinctfrom the corresponding H2 or L2 sequence of the second heterodimer, andwherein H1 and H2 each comprise at least a heavy chain variable domain(V_(H) domain) and a heavy chain constant domain (C_(H1) domain); L1 andL2 each comprise at least a light chain variable domain (V_(L) domain)and a light chain constant domain (C_(L) domain); and at least one ofH1, H2, L1 and L2 comprises at least one amino acid modification of atleast one constant domain and/or at least one variable domain, whereinH1 preferentially pairs with L1 as compared to L2 and H2 preferentiallypairs with L2 as compared to L1.

In some aspects, the construct further comprises a heterodimeric Fc, theFc comprising at least two CH3 sequences, wherein the Fc is coupled,with or without one or more linkers, to the first heterodimer and thesecond heterodimer, wherein the dimerized CH3 sequences have a meltingtemperature (Tm) of about 68° C. or higher as measured by differentialscanning calorimetry (DSC), and wherein the construct is bispecific.

In some aspects, the at least one amino acid modification of is selectedfrom at least one amino acid modification shown in the Tables orExamples.

In some aspects, H1 pairs preferentially with L1 as compared to L2, andH2 pairs preferentially with L2 as compared to L1, when H1, H2, L1 andL2 are co-expressed in a cell or a mammalian cell, or when H1, H2, L1and L2 are co-expressed in a cell-free expression system, or when H1,H2, L1 and L2 are co-produced, or when H1, H2, L1 and L2 are co-producedvia a redox production system.

In some aspects, at least one of H1, H2, L1 and L2 comprises at leastone amino acid modification of a V_(H) and/or V_(L) domain and at leastone amino acid modification of a C_(H1) and/or C_(L) domain such that H1pairs preferentially with L1 as compared to L2, and/or H2 pairspreferentially with L2 as compared to L1.

In some aspects, if H1 comprises at least one amino acid modification inthe C_(H1) domain, then at least one of L1 and L2 comprise at least oneamino acid modification in the C_(L) domain; and/or if H1 comprises atleast one amino acid modification in the V_(H) domain, then at least oneof L1 and L2 comprise at least one amino acid modification in the V_(L)domain.

In some aspects, H1, L1, H2, and/or L2 comprises at least 1, 2, 3, 4, 5,6, 7, 8, 9, or 10 amino acid mutations. In some aspects, at least one ofH1, H2, L1 and L2 comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 aminoacid modifications of at least one constant domain and/or at least onevariable domain.

In some aspects, when both L1 and L2 are co-expressed with at least oneof H1 and H2, the relative pairing of the at least one of H1-L1 andH2-L2 heterodimer pair to that of the respective corresponding H1-L2 orH2-L1 heterodimer pair is greater than 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, or 99%, and wherein the relative pairing of themodified H1-L1 or H2-L2 heterodimer pair is greater than the respectiverelative pairing observed in the corresponding H1-L1 or H2-L2heterodimer pair without the at least one amino acid modification.

In some aspects, the thermal stability as measured by the meltingtemperature (Tm) as measured by DSC of at least one of the first andsecond heterodimers is within about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10°C. of the Tm of the corresponding heterodimer without the at least oneamino acid modification. In some aspects, the thermal stability asmeasured by the melting temperature (Tm) as measured by DSC of eachheterodimer comprising at least one amino acid modification is withinabout 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10° C. of the Tm of thecorresponding heterodimer without the at least one amino acidmodification.

In some aspects, the affinity of each heterodimer for the antigen towhich it binds is within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25,30, 35, 40, 45, or 50-fold of the affinity of the respective unmodifiedheterodimer for the same antigen as measured by surface plasmonresonance (SPR) or FACS.

In some aspects, at least one of H1 and L1 comprises at least one domaincomprising at least one amino acid modification resulting in greatersteric complementarity of amino acids when H1 pairs with L1 as comparedto L2. In some aspects, at least one of H2 and L2 comprises at least onedomain comprising at least one amino acid modification resulting ingreater steric complementarity of amino acids when H2 pairs with L2 ascompared to L1. In some aspects, at least one of H1 and L1 comprises atleast one domain comprising at least one amino acid modificationresulting in greater electrostatic complementarity between charged aminoacids when H1 pairs with L1 as compared to L2. In some aspects, at leastone of H2 and L2 comprises at least one domain comprising at least oneamino acid modification resulting in greater electrostaticcomplementarity between charged amino acids when H2 pairs with L2 ascompared to L1.

In some aspects, the at least one amino acid modification of is a set ofmutations shown in at least one of the Tables or Examples. In someaspects, the at least one modification is not H1-Q39E, L1-Q38K, H2-Q39K,and L2-Q38E. In some aspects, the at least one modification is notH1-Q39E, L1-Q38E, H2-Q39K, and L2-Q38K.

In some aspects, the construct further comprises an Fc comprising atleast two CH3 sequences, wherein the Fc is coupled, with or without oneor more linkers, to the first heterodimer and the second heterodimer.

In some aspects, the Fc is a human Fc, a human IgG1 Fc, a human IgA Fc,a human IgG Fc, a human IgD Fc, a human IgE Fc, a human IgM Fc, a humanIgG2 Fc, a human IgG3 Fc, or a human IgG4 Fc. In some aspects, the Fc isa heterodimeric Fc. In some aspects, the Fc comprises one or moremodifications in at least one of the CH3 sequences. In some aspects, thedimerized CH3 sequences have a melting temperature (Tm) as measured byDSC of about 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 77.5, 78, 79, 80,81, 82, 83, 84, or 85° C. or higher. In some aspects, the Fc is aheterodimer formed with a purity greater than about 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, or 99% when produced; or wherein the Fc is a heterodimer formed witha purity greater than about 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% whenexpressed or when expressed via a single cell. In some aspects, the Fccomprises one or more modifications in at least one of the CH3 sequencesthat promote the formation of a heterodimeric Fc with stabilitycomparable to a wild-type homodimeric Fc. In some aspects, the Fcfurther comprises at least one CH2 sequence. In some aspects, the CH2sequence(s) of the Fc comprises one or more modifications. In someaspects, the Fc comprises one or more modifications to promote selectivebinding of Fc-gamma receptors.

In some aspects, the Fc is coupled to the heterodimers by one or morelinkers, or wherein the Fc is coupled to H1 and H2 by one or morelinkers. In some aspects, the one or more linkers are one or morepolypeptide linkers. In some aspects, the one or more linkers comprisesone or more antibody hinge regions. In some aspects, the one or morelinkers comprises one or more IgG1 hinge regions. In some aspects, theone or more linkers comprises one or more modifications. In someaspects, the one or more modifications to the one or more linkerspromote selective binding of Fc-gamma receptors.

In some aspects, the at least one amino acid modification is at leastone amino acid mutation or wherein the at least one amino acidmodification is at least one amino acid substitution.

In some aspects, the sequences of each of H1, H2, L1, and L2 are derivedfrom human sequences.

In some aspects, the construct is multispecific or bispecific. In someaspects, the construct is multivalent or bivalent.

Also described herein is an isolated polynucleotide or set of isolatedpolynucleotides comprising at least one sequence that encodes aconstruct described herein. In some aspects, the polynucleotide or setof polynucleotides is cDNA.

Also described herein is a vector or set of vectors comprising one ormore of the polynucleotides or sets of polynucleotides described herein.In some aspects, the vector or set of vectors is selected from the groupconsisting of a plasmid, a multi-cistronic vector, a viral vector, anon-episomal mammalian vector, an expression vector, and a recombinantexpression vector.

Also described herein is an isolated cell comprising a polynucleotide orset of polynucleotides described herein or a vector or set of vectorsdescribed herein. In some aspects, the cell is a hybridoma, a ChineseHamster Ovary (CHO) cell, or a HEK293 cell.

Also described herein is a pharmaceutical composition comprising aconstruct described herein and a pharmaceutically acceptable carrier. Insome aspects, the composition further comprises one or more substancesselected from the group consisting of a buffer, an antioxidant, a lowmolecular weight molecule, a drug, a protein, an amino acid, acarbohydrate, a lipid, a chelating agent, a stabilizer, and anexcipient.

Also described herein is a use of a construct described herein or apharmaceutical composition described herein for the treatment of adisease or disorder or cancer or vascular disease in a subject or in themanufacture of a medicine.

Also described herein is a method of treatment of a subject having adisease or disorder or cancer or vascular disease comprisingadministering to the subject a construct described herein or acomposition described herein.

Also described herein is a method of inhibiting, reducing or blocking asignal within or to a cell, comprising contacting the cell with aconstruct described herein or a composition described herein.

Also described herein is a method of obtaining a construct describedherein from a host cell culture, the method comprising the steps of: (a)obtaining a host cell culture comprising at least one host cellcomprising one or more nucleic acid sequences encoding the construct;and (b) recovering the construct from the host cell culture.

Also described herein is a method of obtaining a construct describedherein comprising the steps of: (a) obtaining H1, L1, H2, and L2; (b)allowing H1 to pair preferentially with L1 as compared to L2 and H2 topair preferentially with L2 as compared to L1; and (c) obtaining theconstruct.

Also described herein is a method of preparing a construct describedherein comprising: obtaining a polynucleotide or set of polynucleotidesencoding at least one construct; determining the optimal ratios of eachof the polynucleotide or set of polynucleotides for introduction into atleast one host cell, wherein the optimal ratios are determined byassessing the amount of H1-L1 and H2-L2 heterodimer pairs formed uponexpression of H1, L1, H2, and L2 as compared to mispaired H1-L2 andH2-L1 heterodimer pairs formed upon expression of H1, L1, H2, and L2;selecting a preferred optimal ratio, wherein transfection of at leastone host cell with the preferred optimal ratio of the polynucleotide orset of polynucleotides results in expression of the construct;transfecting the at least one host cell with the optimal ratio of thepolynucleotide or set of polynucleotides; and culturing the at least onehost cell to express the construct.

In some aspects, selecting the optimal ratio is assessed by transfectionin a transient transfection system. In some aspects, transfection of theat least one host cell with the preferred optimal ratio of thepolynucleotide or set of polynucleotides results in optimal expressionof the construct. In some aspects, the construct comprises an Fccomprising at least two CH3 sequences, wherein the Fc is coupled, withor without one or more linkers, to the first heterodimer and the secondheterodimer. In some aspects, the Fc is a heterodimer, optionallycomprising one or more amino acid modifications.

Also described herein is a computer-readable storage medium storing adataset comprising data representing complementary mutations in a firstheterodimer comprising a first immunoglobulin heavy chain polypeptidesequence (H1) and a first immunoglobulin light chain polypeptidesequence (L1); and a second heterodimer comprising a secondimmunoglobulin heavy chain polypeptide sequence (H2) and a secondimmunoglobulin light chain polypeptide sequence (L2), wherein H1 and H2each comprise at least a heavy chain variable domain (V_(H) domain) anda heavy chain constant domain (C_(H1) domain); wherein L1 and L2 eachcomprise at least a light chain variable domain (V_(L) domain) and alight chain constant domain (C_(L) domain), and wherein the dataset ofcomplementary mutations comprises data representing those mutationslisted in the Tables or Examples or a subset of those mutations; andcomputer executable code for determining the likelihood that H1 willpair preferentially with L1 as compared to L2 and/or H2 will pairpreferentially with L2 as compared to L1.

Also described herein is a computer implemented method for determiningpreferential pairing, comprising: obtaining a dataset comprising datarepresenting complementary mutations in a first heterodimer comprising afirst immunoglobulin heavy chain polypeptide sequence (H1) and a firstimmunoglobulin light chain polypeptide sequence (L1); and a secondheterodimer comprising a second immunoglobulin heavy chain polypeptidesequence (H2) and a second immunoglobulin light chain polypeptidesequence (L2), wherein H1 and H2 each comprise at least a heavy chainvariable domain (V_(H) domain) and a heavy chain constant domain (C_(H1)domain); wherein L1 and L2 each comprise at least a light chain variabledomain (V_(L) domain) and a light chain constant domain (C_(L) domain),and wherein the dataset of complementary mutations comprises datarepresenting those mutations listed in the Tables or Examples or asubset of those mutations; and determining, by a computer processor, thelikelihood that H1 will pair preferentially with L1 as compared to L2and/or H2 will pair preferentially with L2 as compared to L1. In someaspects, the method further comprises producing a construct describedherein.

Also described herein is a method of producing a bi-specific antigenbinding polypeptide construct, said bi-specific construct comprising afirst heterodimer comprising a first immunoglobulin heavy chainpolypeptide sequence (H1), and a first immunoglobulin light chainpolypeptide sequence (L1) from a first mono-specific antigen bindingpolypeptide; and a second heterodimer comprising a second immunoglobulinheavy chain polypeptide sequence (H2), and a second immunoglobulin lightchain polypeptide sequence (L2) from a second mono-specific antigenbinding polypeptide, wherein H1 and H2 each comprise at least a heavychain variable domain (V_(H) domain) and a heavy chain constant domain(C_(H1) domain); wherein L1 and L2 each comprise at least a light chainvariable domain (V_(L) domain) and a light chain constant domain (C_(L)domain), the method comprising: obtaining a dataset comprising datarepresenting a set of amino acid modifications within H1, H2, L1 and L2such that upon introduction of a subset of the modifications into H1,H2, L1 and/or L2, H1 pairs preferentially with L1 as compared to L2 andH2 pairs preferentially with L2 as compared to L1 in a test system;introducing a subset of one or more modifications from the dataset intothe first heterodimer and/or the second heterodimer; and co-expressingthe first heterodimer and the second heterodimer in at least one hostcell to produce an expression product comprising the bi-specificconstruct.

In some aspects, the method further comprises determining the amount ofthe bi-specific construct in the expression product relative to otherpolypeptide products. In some aspects, the bi-specific construct isproduced with a purity of greater than 70% compared to the otherpolypeptide products. In some aspects, the dataset is a datasetdescribed herein. In some aspects, the method further comprises the stepof adding additional amino acid modifications to at least one of H1, H2,L1, or L2 to increase the purity of the bi-specific construct comparedto the other polypeptide products. In some aspects, the constructcomprises an Fc comprising at least two CH3 sequences, wherein the Fc iscoupled, with or without one or more linkers, to the first heterodimerand the second heterodimer. In some aspects, the Fc is a heterodimer,optionally comprising one or more amino acid modifications. In someaspects, the antigen binding polypeptide is an antibody, a Fab, or ascFv.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1B provides a Table showing the preferential pairing ofheterodimers, from antigen-binding constructs described herein, in LCCAdesign sets where amino acid modifications have been made to the V_(H)or V_(L) domains.

FIGS. 2A-2B provides a Table showing the preferential pairing ofheterodimers in LCCA design sets where amino acid modifications havebeen made to the C_(H1) or C_(L) domains.

FIG. 3 provides a Table showing the thermal stability and affinity forantigen for selected preferentially paired or mispaired heterodimersfrom antigen-binding constructs described herein.

FIG. 4 provides thermal unfolding curves for selected heterodimers.

FIGS. 5A-5D provides size exclusion chromatography profiles for selectedheterodimers.

FIGS. 6A-E depicts D3H44 heavy chain and light chain amino acidsequences aligned against canonical human germline sequences forVariable, Constant and J-region segments. (Notations in figs: * sequenceidentity, #Interface hotspots (specificity drivers), + mutated residuestested in designs). FIG. 6A depicts Human VH germline subgroups (onerepresentative sequence is displayed for each family). Sequence identitybased on an alignment of D3H44 against VH3 and IGHJ3*02. FIG. 6Adiscloses SEQ ID NOS 20-34, respectively, in order of appearance. FIG.6B depicts Human kappa VL germline subgroups (one representativesequence is displayed from each family). Sequence identity based on analignment of D3H44 against VKI and IGKJ1*01. FIG. 6B discloses SEQ IDNOS 35-47, respectively, in order of appearance. FIG. 6C depicts Humanlambda VL germline subgroups (one representative sequence is displayedfrom each family). Sequence identity based on an alignment of D3H44against VL1 and IGLJ1*01. FIG. 6C discloses SEQ ID NOS 35, 48-57, 42,and 58-64, respectively, in order of appearance. FIG. 6D depicts humanCH1 allele sequences. FIG. 6D discloses SEQ ID NOS 65-74, respectively,in order of appearance. FIG. 6E depicts Human kappa and lambda allelesequences. FIG. 6E discloses SEQ ID NOS 75-80, 75, and 81-85,respectively, in order of appearance.

FIG. 7 depicts a flow chart outlining a strategy for designing abi-specific antibody.

FIG. 8 illustrates a high level schematic overview of the engineeringrequirements for forming a bispecific Mab (monoclonal antibody), and theassay requirements needed to quantify heavy chain light chain pairs. Thedesign goal of engineering a bispecific Mab with high purity (i.e.,little or no mispaired H-L associations) can be achieved by rationallyengineering (via the introduction of specific amino acid mutations) thepreferential pairing of two unique heavy chains for their unique cognatelight chains. This process is shown schematically; here H1 has beenengineered to preferentially pair with L1 and not L2. Likewise, H2 hasbeen engineered to preferentially pair with L2 and not L1. Theexperimental screening of bispecific Mab designs requires an assaycapable of simultaneously quantifying H1-L1:H1-L2 and H2-L2:H2-L1. Theseassay requirements can be simplified by assuming that each bispecificFab arm can be independently engineered. In this case, the assay wouldonly need to quantify H1-L1:H1-L2 or H2-L2:H2-L1, and not bothsimultaneously.

FIG. 9 provides a schematic depicting how heavy chains and light chainsare tagged and preferential pairing is determined. In this schematic,the circle represents a cell in which 3 constructs are transfected. Theexpression products are secreted from the cell and the supernatant(SPNT) is flowed over a detection device, in this case an SPR chip.Based on the detection level of the two different tags fused to the twolight chains competing for heavy chain pairing, a quantitative estimateof the preferential pairing of the heavy chain to the two light chainscan be estimated.

FIG. 10 depicts the heavy chain associated products expected when eachof two full-length heavy chains is independently co-expressed with twodifferent light chains. Preferential pairing is assessed using MCA.

FIG. 11 depicts an exemplary set of H1, L1, H2, L2 chains which havebeen designed such that H1 preferentially pairs with L1 over L2 and H2preferentially pairs with L2 over L1. A cartoon representation of the 3Dcrystal structure of the variable region heavy and light chain interfaceis presented. The mutations introduced at the interface achieveelectrostatic and steric complementarity in the two set of variableregion interface respectively for the preferentially forming obligatepair. On the other hand, there is unfavorable steric and electrostaticmismatch in the incorrect pair that would result in reduced pairingpropensity for the mismatched pair as well as reduced stability.

FIGS. 12A-12B depicts LC/MS spectra resulting when H1, L1 and L2 areco-expressed (left panel) and when H2, L1, and L2 are co-expressed(right panel), based on the exemplary set of H1, L1, H2, L2 chains shownin FIG. 11.

FIGS. 13 A-C depicts assessment of the biophysical properties of theH1-L1 and H2-L2 pairs based on the design shown in FIG. 11. FIG. 13Ashows non-reducing SDS-PAGE analysis of the H1-L1 and H2-L2 pairs beforeand after protein A purification, and the yield of product is shown atthe bottom of the gels; FIG. 13B shows the DSC thermograms of the H1-L1and H2-L2 paired products; and FIG. 13C shows UPLC-SEC (H2-L2) profilefor the H2-L2 pair.

FIG. 14 depicts the heavy chain associated products expected when twodifferent light chains are co-expressed with two different heavy chainsin a cell. Preferential pairing is assessed using an SMCA (monoclonalantibody competition assay).

FIGS. 15A-15C depicts the LC/MS spectra for the bispecificantigen-binding construct (H1-L1_H2-L2) based on design shown in FIG.11.

FIG. 16A Left panel depicts an assessment of the purity of thebispecific antigen-binding construct derived from design set as depictedin FIG. 11. FIG. 16A shows a Coomassie stained non-reducing SDS-PAGE ofthe SMCA variant, along with a control variant composed of Her2 bindingMab with a heterodimeric Fc. The purity of the SMCA variant, afterprotein A (ProtA) purification, is high and is qualitatively equivalentto the control. The estimated post-ProtA yield of the SMCA variant is 20mg/L and is comparable to the control yield of 40 mg/ml. FIG. 16Bdepicts the SEC profile of the aforementioned bispecific construct. Themain peak (>90% total peak area) observed runs at a molecular weight of˜150 KDa and is composed of Mab monomers. The observed minor peak runsat ˜75 KDa and consists of half-antibodies. No significant highermolecular weight peaks (potentially indicative of aggregate species) areobserved.

FIG. 17A Upper panel depicts SPR data showing monospecific binding forTF or Her2 antigen by the bispecific construct based on design setdepicted in FIG. 11 (Note: This SMCA design utilizes the same H1-L1 andH2-L2 MCA designs as depicted in FIG. 11). Lower panel depicts SPR datashowing simultaneous bispecific binding of TF and Her2 antigens by thebispecific antigen-binding construct.

FIG. 17B Upper panel depicts SPR data showing monospecific binding fortissue factor (TF) or Her2 antigen by the bispecific construct based ondesign set depicted in FIG. 11 (Note: This SMCA design utilizes the sameH1-L1 and H2-L2 MCA designs as depicted in FIG. 11). Lower panel depictsSPR data showing simultaneous bispecific binding of TF and Her2 antigensby the bispecific antigen binding construct.

FIG. 18 depicts a flowchart for identifying critical interface residuesand for computational modeling of designs with preferential heavy-lightchain pairing.

FIGS. 19A-19C presents a schematic depicting a method of preparing abi-specific antibody using the library of obligate mutation pairsprovided in this invention.

DETAILED DESCRIPTION

Provided herein are antigen binding polypeptide constructs (alsoreferred to as heterodimer pairs) which can comprise a first heterodimerand a second heterodimer wherein each heterodimer comprises animmunoglobulin heavy chain or fragment thereof and an immunoglobulinlight chain. At least one of the heterodimers can comprise one or moreamino acid modifications in the immunoglobulin heavy chain constantdomain 1 (CH1) and one or more amino acid modifications in theimmunoglobulin light chain constant domain (CL); one or more amino acidmodifications in the immunoglobulin heavy chain variable domain (VH) andone or more amino acid modifications in the immunoglobulin light chainvariable domain (VL); or a combination of the preceding amino acidmodifications to both the constant and variable domains of the heavy andlight chains. The amino acids that are modified are typically part ofthe interface between the light chain and heavy chain and are modifiedto create preferential pairing between each heavy chain and the desiredlight chain such that the heavy chain of the first heterodimerpreferentially pairs with one of the light chains rather than the other.Likewise, the heavy chain of the second heterodimer can preferentiallypair with the second light chain rather than first.

As noted above, specific combinations of the amino acid modificationsdescribed herein promote preferential pairing of heavy chains withspecific light chains, thus enabling bi-specific monoclonal antibody(Mab) expression to occur with negligible or limited mispairing, andminimizing the need to purify the desired heterodimers from undesired,or mispaired products. The heterodimers can exhibit comparable thermalstability to heterodimers that do not include the amino acidmodifications, and can also demonstrate binding affinity for antigenthat is comparable to heterodimers that do not include the amino acidmodifications.

The designs of the first and second heterodimers, can be used to createbi-specific antibodies targeting two different therapeutic targets ortargeting two distinct epitopes (overlapping or non-overlapping) withinthe same antigen.

The invention further provides methods of preparing the heterodimerpairs according to the invention.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which the claimed subject matter belongs. In the event that thereare a plurality of definitions for terms herein, those in this sectionprevail. Where reference is made to a URL or other such identifier oraddress, it is understood that such identifiers can change andparticular information on the internet can come and go, but equivalentinformation can be found by searching the internet. Reference theretoevidences the availability and public dissemination of such information.

It is to be understood that the foregoing general description and thefollowing detailed description are exemplary and explanatory only andare not restrictive of any subject matter claimed. In this application,the use of the singular includes the plural unless specifically statedotherwise.

In the present description, any concentration range, percentage range,ratio range, or integer range is to be understood to include the valueof any integer within the recited range and, when appropriate, fractionsthereof (such as one tenth and one hundredth of an integer), unlessotherwise indicated. As used herein, “about” means±10% of the indicatedrange, value, sequence, or structure, unless otherwise indicated. Itshould be understood that the terms “a” and “an” as used herein refer to“one or more” of the enumerated components unless otherwise indicated ordictated by its context. The use of the alternative (e.g., “or”) shouldbe understood to mean either one, both, or any combination thereof ofthe alternatives. As used herein, the terms “include” and “comprise” areused synonymously. In addition, it should be understood that theindividual single chain polypeptides or immunoglobulin constructsderived from various combinations of the structures and substituentsdescribed herein are disclosed by the present application to the sameextent as if each single chain polypeptide or heterodimer were set forthindividually. Thus, selection of particular components to formindividual single chain polypeptides or heterodimers is within the scopeof the present disclosure

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in the applicationincluding, but not limited to, patents, patent applications, articles,books, manuals, and treatises are hereby expressly incorporated byreference in their entirety for any purpose.

It is to be understood that the methods and compositions describedherein are not limited to the particular methodology, protocols, celllines, constructs, and reagents described herein and as such may vary.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the methods and compositions described herein,which will be limited only by the appended claims.

All publications and patents mentioned herein are incorporated herein byreference in their entirety for the purpose of describing anddisclosing, for example, the constructs and methodologies that aredescribed in the publications, which might be used in connection withthe methods, compositions and compounds described herein. Thepublications discussed herein are provided solely for their disclosureprior to the filing date of the present application. Nothing herein isto be construed as an admission that the inventors described herein arenot entitled to antedate such disclosure by virtue of prior invention orfor any other reason.

In the present application, amino acid names and atom names (e.g. N, O,C, etc.) are used as defined by the Protein DataBank (PDB)(www.pdb.org), which is based on the IUPAC nomenclature (IUPACNomenclature and Symbolism for Amino Acids and Peptides (residue names,atom names etc.), Eur. J. Biochem., 138, 9-37 (1984) together with theircorrections in Eur. J. Biochem., 152, 1 (1985). The term “amino acidresidue” is primarily intended to indicate an amino acid residuecontained in the group consisting of the 20 naturally occurring aminoacids, i.e. alanine (Ala or A), cysteine (Cys or C), aspartic acid (Aspor D), glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Glyor G), histidine (His or H), isoleucine (Ile or I), lysine (Lys or K),leucine (Leu or L), methionine (Met or M), asparagine (Asn or N),proline (Pro or P), glutamine (Gln or Q), arginine (Arg or R), serine(Ser or S), threonine (Thr or T), valine (Val or V), tryptophan (Trp orW), and tyrosine (Tyr or Y) residues.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues.That is, a description directed to a polypeptide applies equally to adescription of a peptide and a description of a protein, and vice versa.The terms apply to naturally occurring amino acid polymers as well asamino acid polymers in which one or more amino acid residues is anon-naturally encoded amino acid. As used herein, the terms encompassamino acid chains of any length, including full length proteins, whereinthe amino acid residues are linked by covalent peptide bonds.

The term “nucleotide sequence” or “nucleic acid sequence” is intended toindicate a consecutive stretch of two or more nucleotide molecules. Thenucleotide sequence may be of genomic, cDNA, RNA, semisynthetic orsynthetic origin, or any combination thereof.

“Cell”, “host cell”, “cell line” and “cell culture” are usedinterchangeably herein and all such terms should be understood toinclude progeny resulting from growth or culturing of a cell.“Transformation” and “transfection” are used interchangeably to refer tothe process of introducing a nucleic acid sequence into a cell.

The term “amino acid” refers to naturally occurring and non-naturallyoccurring amino acids, as well as amino acid analogs and amino acidmimetics that function in a manner similar to the naturally occurringamino acids. Naturally encoded amino acids are the 20 common amino acids(alanine, arginine, asparagine, aspartic acid, cysteine, glutamine,glutamic acid, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, praline, serine, threonine, tryptophan,tyrosine, and valine) and pyrrolysine and selenocysteine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, such as,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (such as, norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Reference to an amino acidincludes, for example, naturally occurring proteogenic L-amino acids;D-amino acids, chemically modified amino acids such as amino acidvariants and derivatives; naturally occurring non-proteogenic aminoacids such as alanine, ornithine, etc.; and chemically synthesizedcompounds having properties known in the art to be characteristic ofamino acids. Examples of non-naturally occurring amino acids include,but are not limited to, □-methyl amino acids (e.g. methyl alanine),D-amino acids, histidine-like amino acids (e.g., 2-amino-histidine,hydroxy-histidine, homohistidine), amino acids having an extra methylenein the side chain (“homo” amino acids), and amino acids in which acarboxylic acid functional group in the side chain is replaced with asulfonic acid group (e.g., cysteic acid). The incorporation ofnon-natural amino acids, including synthetic non-native amino acids,substituted amino acids, or one or more D-amino acids into the proteinsof the present invention may be advantageous in a number of differentways. D-amino acid-containing peptides, etc., exhibit increasedstability in vitro or in vivo compared to L-amino acid-containingcounterparts. Thus, the construction of peptides, etc., incorporatingD-amino acids can be particularly useful when greater intracellularstability is desired or required. More specifically, D-peptides, etc.,are resistant to endogenous peptidases and proteases, thereby providingimproved bioavailability of the molecule, and prolonged lifetimes invivo when such properties are desirable. Additionally, D-peptides, etc.,cannot be processed efficiently for major histocompatibility complexclass II-restricted presentation to T helper cells, and are therefore,less likely to induce humoral immune responses in the whole organism.

Amino acids are referred to herein by either their commonly known threeletter symbols or by the one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission. Nucleotides, likewise, may bereferred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, “conservatively modified variants” refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of ordinary skill inthe art will recognize that each codon in a nucleic acid (except AUG,which is ordinarily the only codon for methionine, and TGG, which isordinarily the only codon for tryptophan) can be modified to yield afunctionally identical molecule. Accordingly, each silent variation of anucleic acid which encodes a polypeptide is implicit in each describedsequence.

As to amino acid sequences, one of ordinary skill in the art willrecognize that individual substitutions, deletions or additions to anucleic acid, peptide, polypeptide, or protein sequence which alters,adds or deletes a single amino acid or a small percentage of amino acidsin the encoded sequence is a “conservatively modified variant” where thealteration results in the deletion of an amino acid, addition of anamino acid, or substitution of an amino acid with a chemically similaramino acid. Conservative substitution tables providing functionallysimilar amino acids are known to those of ordinary skill in the art.Such conservatively modified variants are in addition to and do notexclude polymorphic variants, interspecies homologs, and alleles of theinvention.

Conservative substitution tables providing functionally similar aminoacids are known to those of ordinary skill in the art. The followingeight groups each contain amino acids that are conservativesubstitutions for one another:

1) Alanine (A), Glycine (G);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

7) Serine (S), Threonine (T); and

8) Cysteine (C), Methionine (M)

(see, e.g., Creighton, Proteins: Structures and Molecular Properties (WH Freeman & Co.; 2nd edition (December 1993).

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same. Sequences are“substantially identical” if they have a percentage of amino acidresidues or nucleotides that are the same (i.e., about 50% identity,about 55% identity, 60% identity, about 65%, about 70%, about 75%, about80%, about 85%, about 90%, about 95% or about 99% identity over aspecified region), when compared and aligned for maximum correspondenceover a comparison window, or designated region as measured using one ofthe following sequence comparison algorithms (or other algorithmsavailable to persons of ordinary skill in the art) or by manualalignment and visual inspection. This definition also refers to thecomplement of a test sequence. The identity can exist over a region thatis at least about 50 amino acids or nucleotides in length, or over aregion that is 75-100 amino acids or nucleotides in length, or, wherenot specified, across the entire sequence of a polynucleotide orpolypeptide. A polynucleotide encoding a polypeptide of the presentinvention, including homologs from species other than human, may beobtained by a process comprising the steps of screening a library understringent hybridization conditions with a labeled probe having apolynucleotide sequence of the invention or a fragment thereof, andisolating full-length cDNA and genomic clones containing saidpolynucleotide sequence. Such hybridization techniques are well known tothe skilled artisan.

A derivative, or a variant of a polypeptide is said to share “homology”or be “homologous” with the peptide if the amino acid sequences of thederivative or variant has at least 50% identity with a 100 amino acidsequence from the original peptide. In certain embodiments, thederivative or variant is at least 75% the same as that of either thepeptide or a fragment of the peptide having the same number of aminoacid residues as the derivative. In certain embodiments, the derivativeor variant is at least 85% the same as that of either the peptide or afragment of the peptide having the same number of amino acid residues asthe derivative. In certain embodiments, the amino acid sequence of thederivative is at least 90% the same as the peptide or a fragment of thepeptide having the same number of amino acid residues as the derivative.In some embodiments, the amino acid sequence of the derivative is atleast 95% the same as the peptide or a fragment of the peptide havingthe same number of amino acid residues as the derivative. In certainembodiments, the derivative or variant is at least 99% the same as thatof either the peptide or a fragment of the peptide having the samenumber of amino acid residues as the derivative.

As used herein, an “isolated” polypeptide or construct means a constructor polypeptide that has been identified and separated and/or recoveredfrom a component of its natural cell culture environment. Contaminantcomponents of its natural environment are materials that would typicallyinterfere with diagnostic or therapeutic uses for the heteromultimer,and may include enzymes, hormones, and other proteinaceous ornon-proteinaceous solutes.

In certain embodiments, as used herein, “isolated” antigen-bindingconstructs described herein comprise heterodimer pairs or “isolated”heterodimer pairs that comprise a heterodimer or heterodimer pair thathas been identified and separated and/or recovered from a component ofits natural cell culture environment. Contaminant components of itsnatural environment are materials that would interfere with diagnosticor therapeutic uses for the heterodimer or antigen-binding construct,and may include enzymes, hormones, and other proteinaceous ornon-proteinaceous solutes.

The heterodimers and antigen-binding constructs and heterodimer pairsare generally purified to substantial homogeneity. The phrases“substantially homogeneous”, “substantially homogeneous form” and“substantial homogeneity” are used to indicate that the product issubstantially devoid of by-products originated from undesiredpolypeptide combinations (e.g. homodimers). Expressed in terms ofpurity, substantial homogeneity means that the amount of by-productsdoes not exceed 10%, and preferably is below 5%, more preferably below1%, most preferably below 0.5%, wherein the percentages are by weight.

The phrase “selectively (or specifically) hybridizes to” refers to thebinding, duplexing, or hybridizing of a molecule only to a particularnucleotide sequence under stringent hybridization conditions when thatsequence is present in a complex mixture (including but not limited to,total cellular or library DNA or RNA).

Terms understood by those in the art of antibody technology are eachgiven the meaning acquired in the art, unless expressly defineddifferently herein. Antibodies are known to have variable regions, ahinge region, and constant domains. Immunoglobulin structure andfunction are reviewed, for example, in Harlow et al, Eds., Antibodies: ALaboratory Manual, Chapter 14 (Cold Spring Harbor Laboratory, ColdSpring Harbor, 1988).

As used herein, the terms “antibody” and “immunoglobulin” or “antigenbinding polypeptide” are used interchangeably. An “antigen bindingpolypeptide” refers to a polypeptide substantially encoded by animmunoglobulin gene or immunoglobulin genes, or one or more fragmentsthereof, which specifically bind an analyte (antigen). The recognizedimmunoglobulin genes include the kappa, lambda, alpha, gamma, delta,epsilon and mu constant region genes, as well as the myriadimmunoglobulin variable region genes. Light chains are classified aseither kappa or lambda. Heavy chains are classified as gamma, mu, alpha,delta, or epsilon, which in turn define the immunoglobulin isotypes,IgG, IgM, IgA, IgD, and IgE, respectively. Further, the antibody canbelong to one of a number of subtypes, for instance, the IgG can belongto the IgG1, IgG2, IgG3, or IgG4 subclasses.

An exemplary immunoglobulin (antibody) structural unit is composed oftwo pairs of polypeptide chains, each pair having one “light” (about 25kD) and one “heavy” chain (about 50-70 kD). The term “light chain”includes a full-length light chain and fragments thereof havingsufficient variable region sequence to confer binding specificity. Afull-length light chain includes a variable region domain, VL, and aconstant region domain, CL. The variable region domain of the lightchain is at the amino-terminus of the polypeptide. Light chains includekappa chains and lambda chains. The term “heavy chain” includes afull-length heavy chain and fragments thereof having sufficient variableregion sequence to confer binding specificity. A full-length heavy chainincludes a variable region domain, VH, and three constant regiondomains, CH1, CH2, and CH3. The VH domain is at the amino-terminus ofthe polypeptide, and the CH domains are at the carboxyl-terminus, withthe CH3 being closest to the carboxy-terminus of the polypeptide. Heavychains can be of any isotype, including IgG (including IgG1, IgG2, IgG3and IgG4 subclasses), IgA (including IgA1 and IgA2 subclasses), IgM andIgE. The term “variable region” or “variable domain” refers to a portionof the light and/or heavy chains of an antibody generally responsiblefor antigen recognition, typically including approximately theamino-terminal 120 to 130 amino acids in the heavy chain (VH) and about100 to 110 amino terminal amino acids in the light chain (VL). A“complementarity determining region” or “CDR” is an amino acid sequencethat contributes to antigen binding specificity and affinity.“Framework” regions (FR) can aid in maintaining the proper conformationof the CDRs to promote binding between the antigen binding region and anantigen. Structurally, framework regions can be located in antibodiesbetween CDRs. The variable regions typically exhibit the same generalstructure of relatively conserved framework regions (FR) joined by threehyper variable regions, CDRs. The CDRs from the two chains of each pairtypically are aligned by the framework regions, which can enable bindingto a specific epitope. From N-terminal to C-terminal, both light andheavy chain variable regions typically comprise the domains FR1, CDR1,FR2, CDR2, FR3, CDR3, and FR4. The assignment of amino acids to eachdomain is typically in accordance with the definitions of KabatSequences of Proteins of Immunological Interest (National Institutes ofHealth, Bethesda, Md. (1987 and 1991)), unless stated otherwise. Incertain embodiments, the immunoglobulin constructs comprise at least oneimmunoglobulin domain from IgG, IgM, IgA, IgD, or IgE connected to atherapeutic polypeptide. In some embodiments, the immunoglobulin domaincomprised in an immunoglobulin construct provided herein, is from animmunoglobulin based construct such as a diabody, or a nanobody. Incertain embodiments, the immunoglobulin constructs described hereincomprise at least one immunoglobulin domain from a heavy chain antibodysuch as a camelid antibody. In certain embodiments, the immunoglobulinconstructs provided herein comprise at least one immunoglobulin domainfrom a mammalian antibody such as a bovine antibody, a human antibody, acamelid antibody, a mouse antibody or any chimeric antibody.

A “bi-specific,” “dual-specific” or “bifunctional” antigen bindingprotein or antibody is a hybrid antigen binding protein having twodifferent antigen binding sites. Bispecific antigen binding proteins andantibodies are a species of multispecific antigen binding proteinantibody. The two binding sites of a bispecific antigen binding proteinor antibody will bind to two different epitopes, which can reside on thesame or different molecular targets. A “multispecific antigen bindingprotein” or “multispecific antibody” is one that targets more than oneantigen or epitope. A “bivalent antigen binding protein” or “bivalentantibody” comprises two antigen binding sites. In some instances, thetwo binding sites have the same antigen specificities. Bivalent antigenbinding proteins and bivalent antibodies can be bispecific, see, infra.A bivalent antibody other than a “multispecific” or “multifunctional”antibody, in certain embodiments, typically is understood to have eachof its binding sites identical.

The term “preferential pairing” is used herein to describe the pairingpattern of a first polypeptide with a second polypeptide, e.g., animmunoglobulin heavy chain with an immunoglobulin light chain in theantigen-binding constructs and heterodimer pairs described herein. Assuch, “preferential pairing” refers to the preferred pairing of a firstpolypeptide with a second polypeptide when one or more additional,distinct polypeptides are present at the same time as the pairing occursbetween the first and second polypeptide. Typically preferential pairingoccurs as a result of the modification (e.g., amino acid modification)of one or both of the first and second polypeptide. Typicallypreferential pairing results in the paired first and second polypeptidebeing the most abundant dimer present after pairing occurs. It is knownin the art that an immunoglobulin heavy chain (H1) will if co-expressedwith two different immunoglobulin light chains (L1 and L2),statistically pair equally with both light chains, resulting in anapproximate 50:50 mixture of H1 paired with L1 and H1 paired with L2. Inthis context, “preferential pairing” would occur between, for example,H1 and L1, if the amount of the H1-L1 heavy chain-light chainheterodimer was greater than the amount of the H1-L2 heterodimer when H1is co-expressed with both L1 and L2. Thus, in this case, H1preferentially pairs with L1 relative to L2.

Antibody heavy chains pair with antibody light chains and meet orcontact one another at an “interface.” The “interface” includes one ormore “contact” amino acid residues in a first polypeptide that interactwith one or more “contact” amino acid residues of a second polypeptide.In one context, the term interface can be used to describe the interfaceof the dimerized CH3 domain of an Fc, where the Fc is preferably derivedfrom an IgG antibody such as IgG1 and most preferably a human IgG1antibody.

The antibody heavy chain that is to be associated with an antibody lightchain typically meet or contact each other at an “interface.” Theimmunoglobulin light chain operatively associates with theimmunoglobulin heavy chain via the “interface”. The “interface”comprises those one or more “contact” amino acid residues in theimmunoglobulin heavy chain that interact with one or more “contact”amino acid residues in the interface of the immunoglobulin light chain.As used herein, the interface can comprise the VH and CH1 domains of theimmunoglobulin heavy chain and the VL and CL domains of theimmunoglobulin light chain. The “interface” can be derived from an IgGantibody and most preferably a human IgG1 antibody.

The term “amino acid modifications” as used herein includes, but is notlimited to, amino acid mutations, insertions, deletions, substitutions,chemical modifications, physical modifications, and rearrangements.

Antigen Binding Constructs and Heterodimer Pairs

The antigen-binding constructs described herein can comprise a firstheterodimer and a second heterodimer; each heterodimer obtained bypairing an immunoglobulin heavy chain with an immunoglobulin lightchain. The structure and organization of the constant and variabledomains of immunoglobulin heavy and light chains are well known in theart. Immunoglobulin heavy chains typically comprise one variable (VH)domain, and three constant domains, CH1, CH2, and CH3. Immunoglobulinlight chains typically comprise one variable (VL) domain and oneconstant (CL) domain. Various modifications to these typical formats canbe made.

The antigen-binding constructs and heterodimer pairs described hereincan comprise a first heterodimer and a second heterodimer, eachheterodimer comprising an immunoglobulin/antibody heavy chain orfragment thereof having at least a VH and CH1 domain, and animmunoglobulin/antibody light chain having a VL domain and a CL domain.In one embodiment, both heterodimers of the heterodimer pair andantigen-binding construct comprise a full-length immunoglobulin heavychain. In another embodiment, both heterodimers of the heterodimer pairor antigen-binding construct comprise a fragment of the immunoglobulinheavy chain that includes at least a VH and a CH1 domain. In oneembodiment, both heterodimers of the heterodimer pair comprise an aminoterminal fragment of the immunoglobulin heavy chain that comprises atleast a VH and a CH1 domain. In another embodiment, both heterodimers ofthe heterodimer pair comprise a carboxy terminal fragment of theimmunoglobulin heavy chain that comprises at least a VH and a CH1domain.

Each heterodimer of the heterodimer pair can bind specifically to anantigen or epitope. In one embodiment, the immunoglobulin heavy chainand the immunoglobulin light chain of each heterodimer is derived orengineered from a known therapeutic antibody. A therapeutic antibody isone that is effective in treating a disease or disorder in a mammal withor predisposed to the disease or disorder. Suitable therapeuticantibodies from which each heterodimer can be derived include, but arenot limited to abagovomab, adalimumab, alemtuzumab, aurograb,bapineuzumab, basiliximab, belimumab, bevacizumab, briakinumab,canakinumab, catumaxomab, certolizumab pegol, cetuximab, daclizumab,denosumab, efalizumab, galiximab, gemtuzumab ozogamicin, golimumab,ibritumomab tiuxetan, infliximab, ipilimumab, lumiliximab, mepolizumab,motavizumab, muromonab, mycograb, natalizumab, nimotuzumab, ocrelizumab,ofatumumab, omalizumab, palivizumab, panitumumab, pertuzumab,ranibizumab, reslizumab, rituximab, teplizumab, tocilizumab/atlizumab,tositumomab, trastuzumab, PROXINIUM™, RENCAREX™, ustekinumab, andzalutumumab.

In one embodiment, the immunoglobulin heavy chain and the immunoglobulinlight chain of each heterodimer are derived or engineered from anantibody that binds a molecule including, but not limited to, thefollowing list of proteins, as well as subunits, domains, motifs andepitopes belonging to the following list of proteins: renin; a growthhormone, including human growth hormone and bovine growth hormone;growth hormone releasing factor; parathyroid hormone; thyroidstimulating hormone; lipoproteins; alpha-1-antitrypsin; insulin A-chain;insulin B-chain; proinsulin; follicle stimulating hormone; calcitonin;luteinizing hormone; glucagon; clotting factors such as factor VII,factor VIIIC, factor IX, tissue factor (TF), and von Willebrands factor;anti-clotting factors such as Protein C; atrial natriuretic factor; lungsurfactant; a plasminogen activator, such as urokinase or human urine ortissue-type plasminogen activator (t-PA); bombesin; thrombin;hemopoietic growth factor; tumor necrosis factor-alpha and -beta;enkephalinase; RANTES (regulated on activation normally T-cell expressedand secreted); human macrophage inflammatory protein (MIP-1-alpha); aserum albumin such as human serum albumin; Muellerian-inhibitingsubstance; relaxin A-chain; relaxin B-chain; prorelaxin; mousegonadotropin-associated peptide; a microbial protein, such asbeta-lactamase; DNase; IgE; a cytotoxic T-lymphocyte associated antigen(CTLA), such as CTLA-4; inhibin; activin; vascular endothelial growthfactor (VEGF); receptors for hormones or growth factors such as, forexample, EGFR, VEGFR; interferons such as alpha interferon (alpha-IFN),beta interferon (beta-IFN) and gamma interferon (gamma-IFN); protein Aor D; rheumatoid factors; a neurotrophic factor such as bone-derivedneurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4,NT-5, or NT-6), or a nerve growth factor; platelet-derived growth factor(PDGF); fibroblast growth factor such as AFGF and PFGF; epidermal growthfactor (EGF); transforming growth factor (TGF) such as TGF-alpha andTGF-beta, including TGF-1, TGF-2, TGF-3, TGF-4, or TGF-5; insulin-likegrowth factor-I and -II (IGF-I and IGF-II); des (1-3)-IGF-I (brainIGF-I), insulin-like growth factor binding proteins; CD proteins such asCD2, CD3, CD4, CD8, CD11a, CD14, CD18, CD19, CD20, CD22, CD23, CD25,CD33, CD34, CD40, CD40L, CD52, CD63, CD64, CD80 and CD147;erythropoietin; osteoinductive factors; immunotoxins; a bonemorphogenetic protein (BMP); an interferon such as interferon-alpha,-beta, and -gamma; colony stimulating factors (CSFs), such as M-CSF,GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1 to IL-13; TNF-alpha,superoxide dismutase; T-cell receptors; surface membrane proteins; decayaccelerating factor; viral antigen such as, for example, a portion ofthe AIDS envelope, e.g., gp120; transport proteins; homing receptors;addressins; regulatory proteins; cell adhesion molecules such as LFA-1,Mac 1, p150.95, VLA-4, ICAM-1, ICAM-3 and VCAM, a4/p7 integrin, and(Xv/p3 integrin including either a or subunits thereof, integrin alphasubunits such as CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, alpha7,alpha8, alpha9, alphaD, CD11a, CD11b, CD51, CD11c, CD41, alphaIIb,alphaIELb; integrin beta subunits such as, CD29, CD 18, CD61, CD104,beta5, beta6, beta7 and beta8; Integrin subunit combinations includingbut not limited to, alphaVbeta3, alphaVbeta5 and alpha4beta7; a memberof an apoptosis pathway; IgE; blood group antigens; flk2/flt3 receptor;obesity (OB) receptor; mp1 receptor; CTLA-4; protein C; an Eph receptorsuch as EphA2, EphA4, EphB2, etc.; a Human Leukocyte Antigen (HLA) suchas HLA-DR; complement proteins such as complement receptor CR1, C1Rq andother complement factors such as C3, and C5; a glycoprotein receptorsuch as GpIb.alpha., GPIIb/IIIa and CD200; and fragments of any of theabove-listed polypeptides.

In an embodiment, the immunoglobulin heavy and light chains of eachheterodimer are derived or engineered from antibodies that specificallybind cancer antigens including, but not limited to, ALK receptor(pleiotrophin receptor), pleiotrophin, KS 1/4 pan-carcinoma antigen;ovarian carcinoma antigen (CA125); prostatic acid phosphate; prostatespecific antigen (PSA); melanoma-associated antigen p97; melanomaantigen gp75; high molecular weight melanoma antigen (HMW-MAA); prostatespecific membrane antigen; carcinoembryonic antigen (CEA); polymorphicepithelial mucin antigen; human milk fat globule antigen; colorectaltumor-associated antigens such as: CEA, TAG-72, CO17-1A, GICA 19-9,CTA-1 and LEA; Burkitt's lymphoma antigen-38.13; CD19; human B-lymphomaantigen-CD20; CD33; melanoma specific antigens such as ganglioside GD2,ganglioside GD3, ganglioside GM2 and ganglioside GM3; tumor-specifictransplantation type cell-surface antigen (TSTA); virally-induced tumorantigens including T-antigen, DNA tumor viruses and Envelope antigens ofRNA tumor viruses; oncofetal antigen-alpha-fetoprotein such as CEA ofcolon, 514 oncofetal trophoblast glycoprotein and bladder tumoroncofetal antigen; differentiation antigen such as human lung carcinomaantigens L6 and L20; antigens of fibrosarcoma; human leukemia T cellantigen-Gp37; neoglycoprotein; sphingolipids; breast cancer antigenssuch as EGFR (Epidermal growth factor receptor); NY-BR-16; NY-BR-16 andHER2 antigen (p185HER2); polymorphic epithelial mucin (PEM); malignanthuman lymphocyte antigen-APO-1; differentiation antigen such as Iantigen found in fetal erythrocytes; primary endoderm I antigen found inadult erythrocytes; preimplantation embryos; I(Ma) found in gastricadenocarcinomas; M18, M39 found in breast epithelium; SSEA-1 found inmyeloid cells; VEP8; VEP9; Myl; Va4-D5; D156-22 found in colorectalcancer; TRA-1-85 (blood group H); SCP-1 found in testis and ovariancancer; C14 found in colonic adenocarcinoma; F3 found in lungadenocarcinoma; AH6 found in gastric cancer; Y hapten; Ley found inembryonal carcinoma cells; TL5 (blood group A); EGF receptor found inA431 cells; E1 series (blood group B) found in pancreatic cancer; FC10.2found in embryonal carcinoma cells; gastric adenocarcinoma antigen;CO-514 (blood group Lea) found in Adenocarcinoma; NS-10 found inadenocarcinomas; CO-43 (blood group Leb); G49 found in EGF receptor ofA431 cells; MH2 (blood group ALeb/Ley) found in colonic adenocarcinoma;19.9 found in colon cancer; gastric cancer mucins; T5A7 found in myeloidcells; R24 found in melanoma; 4.2, GD3, D1.1, OFA-1, GM2, OFA-2, GD2,and M1:22:25:8 found in embryonal carcinoma cells and SSEA-3 and SSEA-4found in 4 to 8-cell stage embryos; Cutaneous Tcell Lymphoma antigen;MART-1 antigen; Sialy Tn (STn) antigen; Colon cancer antigen NY-CO-45;Lung cancer antigen NY-LU-12 valiant A; Adenocarcinoma antigen ART1;Paraneoplastic associated brain-testis-cancer antigen (onconeuronalantigen MA2; paraneoplastic neuronal antigen); Neuro-oncological ventralantigen 2 (NOVA2); Hepatocellular carcinoma antigen gene 520;TUMOR-ASSOCIATED ANTIGEN CO-029; Tumor-associated antigens MAGE-C1(cancer/testis antigen CT7), MAGE-B1 (MAGE-XP antigen), MAGE-B2 (DAM6),MAGE-2, MAGE-4-a, MAGE-4-b and MAGE-X2; Cancer-Testis Antigen (NY-EOS-1)and fragments of any of the above-listed polypeptides.

Human antibodies can be grouped into isotypes including IgG, IgA, IgE,IgM, and IgD. In one embodiment, an Fc is derived from an IgG isotype.In another embodiment, an Fc is derived from an IgA isotype. In anotherembodiment, an Fc is derived from an IgE isotype. In another embodiment,an Fc is derived from an IgM isotype. In another embodiment, an Fc isderived from an IgD isotype.

Human IgG antibodies can also be divided into the subclasses IgG1, IgG2,IgG3, and IgG4. Thus, in some embodiments, it is contemplated an Fc canbe derived from an IgG1, IgG2, IgG3, or IgG4 subclass of antibodies.

Each heterodimer of the heterodimer pair can bind specifically to anepitope or antigen. In one embodiment, each heterodimer of theheterodimer pair binds to the same epitope. In another embodiment, thefirst heterodimer of the heterodimer pair specifically binds to anepitope on one antigen and the second heterodimer of the heterodimerpair binds specifically to a different epitope on the same antigen. Inanother embodiment, the first heterodimer of the heterodimer pairspecifically binds to an epitope on a first antigen, and the secondheterodimer of the heterodimer pair specifically binds to an epitope ona second antigen that is different from the first antigen. For example,in one embodiment, the first heterodimer binds specifically to TissueFactor, while the second heterodimer binds specifically to antigen Her2(ErbB2). In another embodiment, the first heterodimer binds specificallyto a molecule or cancer antigen described above. In another embodiment,the second heterodimer binds specifically to a molecule or cancerantigen described above. In yet another embodiment, the firstheterodimer binds specifically to antigen CD3, while the secondheterodimer binds specifically to antigen CD19.

As indicated above, in some embodiments, the immunoglobulin heavy chainand the immunoglobulin light chain of each heterodimer can be derived orengineered from a known therapeutic antibody, or from an antibody thatbinds various target molecules or cancer antigens. The amino acid andnucleotide sequences of numerous such molecules are readily available(see for example, GenBank: AJ308087.1 (Humanized anti-human tissuefactor antibody D3H44 light chain variable region and CL domain);GenBank. AJ308086.1 (humanized anti-human tissue factor antibody D3H44heavy chain variable region and CH1 domain); GenBank: HC359025.1(Pertuzumab Fab light chain gene module); GenBank: HC359024.1(Pertuzumab Fab heavy chain gene module); GenBank: GM685465.1 (AntibodyTrastuzumab (=Herceptin)—wildtype; light chain); GenBank: GM685463.1(Antibody Trastuzumab (=Herceptin)—wildtype; heavy chain); GenBank:GM685466.1 (Antibody Trastuzumab (=Herceptin)—GC-optimized light chain);and GenBank: GM685464.1 (Antibody Trastuzumab (=Herceptin)—GC-optimizedheavy chain. The sequences of each of the GenBank numbers describedherein are available from the NCBI website as of Nov. 28, 2012 and areeach incorporated by reference in its entirety for all purposes.

In some aspects, an isolated antigen-binding construct comprises anamino acids sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, or 100% identical to an amino acid sequence or fragmentthereof set forth in the Tables or accession numbers disclosed herein.In some aspects, an isolated antigen-binding construct comprises anamino acids sequence encoded by a polynucleotide that is at least 80,85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to anucleotide sequence or fragment thereof set forth in Tables or accessionnumbers disclosed herein.

Amino Acid Modifications to Immunoglobulin Heavy and Light Chains

At least one of the heterodimers of a heterodimer pair can comprise oneor more amino acid modifications to their immunoglobulin heavy and/orimmunoglobulin light chains such that the heavy chain of the firstheterodimer preferentially pairs with one of the light chains ratherthan the other. Likewise, the heavy chain of the second heterodimer canpreferentially pair with the second light chain rather than the first.This preferential pairing of one heavy chain with one of two lightchains can be based on design sets comprising one immunoglobulin heavychain and two immunoglobulin light chains where the immunoglobulin heavychain preferentially pairs with one of the two immunoglobulin lightchains over the other when the immunoglobulin heavy chain isco-expressed with both immunoglobulin light chains. Thus, a LCCA designset can comprise one immunoglobulin heavy chain, a first immunoglobulinlight chain and a second immunoglobulin light chain.

In one embodiment, the one or more amino acid modifications comprise oneor more amino acid substitutions.

In one embodiment, the preferential pairing demonstrated in the LCCAdesign set is established by modifying one or more amino acids that arepart of the interface between the light chain and heavy chain. In oneembodiment, the preferential pairing demonstrated in the LCCA design setis established by modifying one or more amino acids in at least one ofthe CH1 domain of the immunoglobulin heavy chain, the CL domain of afirst immunoglobulin light chain and the CL domain of the secondimmunoglobulin light chain.

In one embodiment the one or amino acid modifications are limited to theconserved framework residues of the variable (VH, VL) and constant (CH1,CL) domains as indicated by the Kabat numbering of residues. Forexample, Almagro [Frontiers In Bioscience (2008) 13: 1619-1633] providesa definition of the framework residues on the basis of Kabat, Chotia,and IMGT numbering schemes.

In one embodiment, at least one of the heterodimers comprises one ormore mutations introduced in the immunoglobulin heavy and immunoglobulinlight chains that are complementary to each other. Complementarity atthe heavy and light chain interface can be achieved on the basis ofsteric and hydrophobic contacts, electrostatic/charge interactions or acombination of the variety of interactions. The complementarity betweenprotein surfaces is broadly described in the literature in terms of lockand key fit, knob into hole, protrusion and cavity, donor and acceptoretc., all implying the nature of structural and chemical match betweenthe two interacting surfaces. In one embodiment, at least one of theheterodimers comprises one or more mutations where the mutationsintroduced in the immunoglobulin heavy and immunoglobulin light chainsintroduce a new hydrogen bond across the light and heavy chain at theinterface. In one embodiment, at least one of the heterodimers comprisesone or more mutations where the mutations introduced in theimmunoglobulin heavy and immunoglobulin light chains introduce a newsalt bridge across the light and heavy chain at the interface.

Non-limiting examples of suitable LCCA design sets are shown in Table 1,showing amino acid modifications in one immunoglobulin heavy chain CH1domain (H1) and the two immunoglobulin light chain CL domains (L1 andL2) of the heterodimers, where H1 preferentially pairs with L1 when H1,L1 and L2 are co-expressed. The amino acid modifications shown in theseLCCA design sets are based on the amino acid sequence of anti-tissuefactor antibody D3H44 immunoglobulin heavy and light chains.

TABLE 1 Selected LCCA design sets with constant domain modifications toone immunoglobulin heavy chain (H1) and two immunoglobulin light chains,L1 and L2, where H1 preferentially pairs with L1 Set # H1_mutation*L1_mutation L2_mutation C500 WT# WT F116A C503 WT WT F98L C505A139W_V190S F116S F118W_V133S C507 A139W_V190S F116A F118W_V133S C509A139W WT F116A C510 A139V_V190S F116A F118W_V133S C511 A139V_V190S WTF118W_V133S C513 A139I_V190S F116A F118W_V133S C514 A139I_V190S WTF118W_V133S C515 A139G_V190A L135W_N137A F116A_L135A C517 A139G_V190AL135W F116A_L135A C519 A139G_V190A L135W F116A_L135V C521 S188I WTS176V_T178L C523 V190G F116A F118W_V133S C524 V190G F116S F118W_V133SC525 S188L_V190Y V133S S176L C527 F174V_P175S_S188G S176L WT C530D146G_Q179R Q124E_Q160E_T178D Q160K_T178R C532 L143A_D144G_Q179RQ124E_V133W_Q160E_T180E V133A_Q160K_T178R *Kabat numbering; #WT refersto a wild-type immunoglobulin chain without amino acid mutations

Additional non-limiting examples of suitable LCCA design sets are shownin Table 2, showing amino acid modifications in one immunoglobulin heavychain CH1 domain (H2) and the two immunoglobulin light chain CL domains(L1 and L2) of the heterodimers, where H2 preferentially pairs with L2when H2, L1 and L2 are co-expressed:

TABLE 2 Selected LCCA design sets with constant domain modifications toone immunoglobulin heavy chain (H2) and two immunoglobulin light chains,L1 and L2, where H2 preferentially pairs with L2 Set # H2_mutation*L1_mutation L2_mutation C501 A139W_V190S WT# F116A C502 A139W_V190A WTF116A C504 F100W WT F98L C506 A139W F116S F118W_V133S C508 A139V F116AF118W_V133S C512 A139I WT F118W_V133S C516 A139W L135W_N137A F116A_L135AC518 A139W L135W F116A_L135A C520 A139W L135W F116A_L135V C522 WT WTS176V_T178L C526 F174V_P175S_S188G V133S S176L C528 F174V_S188L S176L WTC529 S188L S176L WT C531 K145T_Q179D_S188L Q124E_Q160E_T178D Q160K_T178RC533 K145T_Q179D_S188F Q124E_V133W_Q160E_T180E V133A_Q160K_T178R *Kabatnumbering; #WT refers to a wild-type immunoglobulin chain without aminoacid mutations

Additional non-limiting examples of suitable LCCA design sets aredescribed in the Examples, Tables, and Figs.

In one embodiment, the LCCA design sets comprises an immunoglobulinheavy chain with at least one amino acid modification in the CH1 domain,a first immunoglobulin light chain with at least one amino acidmodification in the CL domain, and a second immunoglobulin light chainwithout any amino acid modifications in the CL domain. In anotherembodiment, the LCCA design set comprises an immunoglobulin heavy chainwith at least one amino acid modification in the CH1 domain, a firstimmunoglobulin light chain with at least one amino acid modification inthe CL domain, and a second immunoglobulin light chain with at least oneamino acid modification in the CL domain. In another embodiment, theLCCA design set comprises an immunoglobulin heavy chain with at leastone amino acid modification in the CH1 domain, a first immunoglobulinlight chain with at least two amino acid modifications in the CL domain,and a second immunoglobulin light chain with at least two amino acidmodifications in the CL domain. In another embodiment, the LCCA designset comprises an immunoglobulin heavy chain with at least one amino acidmodification in the CH1 domain, a first immunoglobulin light chain withat least two amino acid modifications in the CL domain, and a secondimmunoglobulin light chain with at least one amino acid modification inthe CL domain.

In one embodiment, the LCCA design set comprises an immunoglobulin heavychain with no amino acid modifications in the CH1 domain, a firstimmunoglobulin light chain with no amino acid modifications in the CLdomain, and a second immunoglobulin light chain with at least one aminoacid modification in the CL domain. In one embodiment, the LCCA designset comprises an immunoglobulin heavy chain with no amino acidmodifications in the CH1 domain, a first immunoglobulin light chain withno amino acid modifications in the CL domain, and a secondimmunoglobulin light chain with at least two amino acid modifications inthe CL domain.

In one embodiment, the LCCA design set comprises an immunoglobulin heavychain with at least two amino acid modifications in the CH1 domain, afirst immunoglobulin light chain with no amino acid modifications in theCL domain, and a second immunoglobulin light chain with at least oneamino acid modification in the CL domain. In one embodiment, the LCCAdesign set comprises an immunoglobulin heavy chain with at least twoamino acid modifications in the CH1 domain, a first immunoglobulin lightchain with at least one amino acid modifications in the CL domain, and asecond immunoglobulin light chain with at least one amino acidmodification in the CL domain. In one embodiment, the LCCA design setcomprises an immunoglobulin heavy chain with at least two amino acidmodifications in the CH1 domain, a first immunoglobulin light chain withat least one amino acid modification in the CL domain, and a secondimmunoglobulin light chain with at least two amino acid modifications inthe CL domain. In one embodiment, the LCCA design set comprises animmunoglobulin heavy chain with at least two amino acid modifications inthe CH1 domain, a first immunoglobulin light chain with at least twoamino acid modifications in the CL domain, and a second immunoglobulinlight chain with at least two amino acid modifications in the CL domain.In one embodiment, the LCCA design set comprises an immunoglobulin heavychain with at least two amino acid modifications in the CH1 domain, afirst immunoglobulin light chain with at least three amino acidmodifications in the CL domain, and a second immunoglobulin light chainwith at least two amino acid modifications in the CL domain.

In one embodiment, the LCCA design set comprises an immunoglobulin heavychain with at least three amino acid modifications in the CH1 domain, afirst immunoglobulin light chain with no amino acid modifications in theCL domain, and a second immunoglobulin light chain with at least oneamino acid modifications in the CL domain. In one embodiment, the LCCAdesign set comprises an immunoglobulin heavy chain with at least threeamino acid modifications in the CH1 domain, a first immunoglobulin lightchain with at least one amino acid modification in the CL domain, and asecond immunoglobulin light chain with at least one amino acidmodification in the CL domain. In one embodiment, the LCCA design setcomprises an immunoglobulin heavy chain with at least three amino acidmodifications in the CH1 domain, a first immunoglobulin light chain withat least three amino acid modifications in the CL domain, and a secondimmunoglobulin light chain with at least two amino acid modifications inthe CL domain. In one embodiment, the LCCA design set comprises animmunoglobulin heavy chain with at least three amino acid modificationsin the CH1 domain, a first immunoglobulin light chain with at least fouramino acid modifications in the CL domain, and a second immunoglobulinlight chain with at least three amino acid modifications in the CLdomain.

In one embodiment, the preferential pairing demonstrated in the LCCAdesign set is established by modifying one or more amino acids in atleast one of the VH domain of the immunoglobulin heavy chain, the VLdomain of a first immunoglobulin light chain and the VL domain of thesecond immunoglobulin light chain. Non-limiting examples of suitableLCCA design sets are shown in Table 3, showing amino acid modificationsin one immunoglobulin heavy chain VH domain (H1) and the twoimmunoglobulin light chain VL domains (L1 and L2) of the heterodimers,where H1 preferentially pairs with L1 when H1, L1 and L2 areco-expressed:

TABLE 3 Selected LCCA design sets with variable domain modifications toone immunoglobulin heavy chain and two immunoglobulin light chains,where H1 preferentially pairs with L1 Set # H1_mutation* L1_mutationL2_mutation V001 V37W_W103H F98L F98W V004 V37W_W103H F98L P44W V005V37A_W103H P44W F98L V006 V37W_W103F F98L F98W V007 V37W F98A F98W V009V37W F98A WT# V011 V37I WT F98L V013 V37A_W103V P44W F98A V015V37A_W103H P44W F98A V016 V37A_W103H P44W F98W V020 L45W Y87G P44W V022WT F98W F98A V023 WT WT F98A V024 Q39R Q38E F98A V026 Q39R Q38E WT V028Q39R Q38E Q38R V030 Q39R Q38D Q38R V032 Q39M Q38M Q38E V034 Q39KQ38N_T85E Q38N_T85K V037 Q39E Q38R F98A V039 Q39D Q38R Q38D V040 V37EL89R_F98T WT V042 V37E_F100D L89R_F98W WT V044 V37E_F100D L89R_F98W F98Y*Kabat numbering; #WT refers to a wild-type immunoglobulin chain withoutamino acid mutations

Additional non-limiting examples of suitable LCCA design sets aredepicted in Table 4, showing amino acid modifications in oneimmunoglobulin heavy chain VH domain (H2) and the two immunoglobulinlight chain VL domains (L1 and L2) of the heterodimers, where H2preferentially pairs with L2 when H2, L1 and L2 are co-expressed:

TABLE 4 Selected LCCA design sets with variable domain modifications toone immunoglobulin heavy chain and two immunoglobulin light chains,where H2 preferentially pairs with L2 Set # H2_mutation* L1_mutationL2_mutation V002 V37I F98L F98W V003 WT# F98L F98W V005 V37A_W103H F98LP44W V007 V37W F98W F98A V008 V37I F98A F98W V009 V37W WT F98A V010 V37IF98A WT V012 F100W WT F98L V014 V37W P44W F98A V017 V37A_W103V P44W F98WV018 V37A_W103V P44W F98L V019 V37I_F100W P44W F98L V021 V37A_W103H Y87GP44W V025 V37W Q38E F98A V027 WT Q38E WT V029 Q39E Q38E Q38R V030 Q39RQ38R Q38D V031 Q39E Q38D Q38R V033 Q39R Q38M Q38E V035 Q39D Q38N_T85EQ38N_T85K V036 Q39E Q38N_T85E Q38N_T85K V038 V37W Q38R F98A V041 WTL89R_F98T WT V043 WT L89R_F98W WT V045 V37S_A97K L89R_F98W F98Y *Kabatnumbering; #WT refers to a wild-type immunoglobulin chain without aminoacid mutations

In one embodiment, the LCCA design set comprises an immunoglobulin heavychain with no amino acid modifications in the VH domain, a firstimmunoglobulin light chain with no amino acid modifications in the VLdomain, and a second immunoglobulin light chain with at least one aminoacid modification in the VL domain. In one embodiment, the LCCA designset comprises an immunoglobulin heavy chain with no amino acidmodifications in the VH domain, a first immunoglobulin light chain withno amino acid modifications in the VL domain, and a secondimmunoglobulin light chain with at least two amino acid modifications inthe VL domain.

In one embodiment, the LCCA design set comprises an immunoglobulin heavychain with at least one amino acid modification in the VH domain, afirst immunoglobulin light chain with no amino acid modifications in theVL domain, and a second immunoglobulin light chain with at least oneamino acid modification in the VL domain. In one embodiment, the LCCAdesign set comprises an immunoglobulin heavy chain with at least oneamino acid modification in the VH domain, a first immunoglobulin lightchain with at least one amino acid modification in the VL domain, and asecond immunoglobulin light chain with at least one amino acidmodification in the VL domain. In one embodiment, the LCCA design setcomprises an immunoglobulin heavy chain with at least one amino acidmodification in the VH domain, a first immunoglobulin light chain withat least two amino acid modifications in the VL domain, and a secondimmunoglobulin light chain with at least two amino acid modifications inthe VL domain.

In one embodiment, the LCCA design set comprises an immunoglobulin heavychain with at least two amino acid modifications in the VH domain, afirst immunoglobulin light chain with no amino acid modifications in theVL domain, and a second immunoglobulin light chain with at least oneamino acid modification in the VL domain. In one embodiment, the LCCAdesign set comprises an immunoglobulin heavy chain with at least twoamino acid modifications in the VH domain, a first immunoglobulin lightchain with at least two amino acid modifications in the VL domain, and asecond immunoglobulin light chain with at least one amino acidmodification in the VL domain. In one embodiment, the LCCA design setcomprises an immunoglobulin heavy chain with at least two amino acidmodifications in the VH domain, a first immunoglobulin light chain withat least one amino acid modification in the VL domain, and a secondimmunoglobulin light chain with at least one amino acid modification inthe VL domain.

In one embodiment, the LCCA design sets shown in Tables 1 to 4 arecombined to provide a combination comprising two distinct immunoglobulinheavy chains (H1 and H2) and two distinct immunoglobulin light chains(L1 and L2), where H1 preferentially pairs with L1 and H2 preferentiallypairs with L2 when H1, H2, L1, and L2 are co-expressed. In oneembodiment, a LCCA design set from Table 1, comprising modifications tothe CH1 domain of the heavy chain and/or the CL domain of the lightchains is combined with a LCCA design set from Table 2, also comprisingmodifications to the CH1 domain of the heavy chain and/or the CL domainof the light chains.

Non-limiting examples of design sets derived from combinations of LCCAdesign sets are shown in Table 5:

TABLE 5 Design sets comprising constant domain modifications Set # Set #H1_mutation* L1_mutation H2_mutation L2_mutation C500 C501 WT# WTA139W_V190S F116A C500 C502 WT WT A139W_V190A F116A C503 C504 WT WTF100W F98L C505 C506 A139W_V190S F116S A139W F118W_V133S C507 C508A139W_V190S F116A A139V F118W_V133S C509 C501 A139W WT A139W_V190S F116AC509 C502 A139W WT A139W_V190A F116A C510 C508 A139V_V190S F116A A139VF118W_V133S C511 C512 A139V_V190S WT A139I F118W_V133S C513 C508A139I_V190S F116A A139V F118W_V133S C514 C512 A139I_V190S WT A139IF118W_V133S C515 C516 A139G_V190A L135W_N137A A139W F116A_L135A C517C518 A139G_V190A L135W A139W F116A_L135A C519 C520 A139G_V190A L135WA139W F116A_L135V C521 C522 S188I WT WT S176V_T178L C523 C508 V190GF116A A139V F118W_V133S C524 C506 V190G F116S A139W F118W_V133S C525C526 S188L_V190Y V133S F174V_P175S_S188G S176L C527 C528F174V_P175S_S188G S176L F174V_S188L WT C527 C529 F174V_P175S_S188G S176LS188L WT C530 C531 D146G_Q179R Q124E_Q160E_T178D K145T_Q179D_S188LQ160K_T178R C532 C533 L143A_D144G_Q179R Q124E_V133W_Q160E_T180EK145T_Q179D_S188F V133A_Q160K_T178R *Kabat numbering; #WT refers to awild-type immunoglobulin chain without amino acid mutations

In one embodiment, a LCCA design set from Table 3, comprisingmodifications to the VH domain of the heavy chain and/or the VL domainof the light chains is combined with a LCCA design set from Table 4,also comprising modifications to the VH domain of the heavy chain and/orthe VL domain of the light chains. Non-limiting examples of design setsderived from such combinations of LCCA design sets are shown in Table 6:

TABLE 6 Design sets comprising variable domain modifications Set # Set #H1_mutation* L1_mutation H2_mutation L2_mutation V001 V002 V37W_W103HF98L V37I F98W V001 V003 V37W_W103H F98L WT# F98W V004 V005 V37W_W103HF98L V37A_W103H P44W V006 V002 V37W_W103F F98L V37I F98W V006 V003V37W_W103F F98L WT F98W V007 V008 V37W F98A V37I F98W V009 V010 V37WF98A V37I WT V011 V012 V37I WT F100W F98L V013 V014 V37A_W103V P44W V37WF98A V015 V014 V37A_W103H P44W V37W F98A V016 V017 V37A_W103H P44WV37A_W103V F98W V005 V018 V37A_W103H P44W V37A_W103V F98L V005 V019V37A_W103H P44W V37I_F100W F98L V020 V021 L45W Y87G V37A_W103H P44W V022V007 WT F98W V37W F98A V023 V009 WT WT V37W F98A V024 V025 Q39R Q38EV37W F98A V026 V027 Q39R Q38E WT WT V028 V029 Q39R Q38E Q39E Q38R V030V031 Q39R Q38D Q39E Q38R V032 V033 Q39M Q38M Q39R Q38E V034 V035 Q39KQ38N_T85E Q39D Q38N_T85K V034 V036 Q39K Q38N_T85E Q39E Q38N_T85K V037V038 Q39E Q38R V37W F98A V039 V030 Q39D Q38R Q39R Q38D V040 V041 V37EL89R_F98T WT WT V042 V043 V37E_F100D L89R_F98W WT WT V044 V045V37E_F100D L89R_F98W V37S_A97K F98Y *Kabat numbering; #WT refers to awild-type immunoglobulin chain without amino acid mutations

Transferability of Specific Amino Acid Modifications Identified Hereinto Other Antibodies:

Although the specific amino acid modifications to immunoglobulin heavyand light chains identified above have been described with respect tothe D3H44 anti-tissue factor extracellular domain antibodyimmunoglobulin heavy and light chains, it is contemplated anddemonstrated herein (see Examples, Figs, and Tables) that these aminoacid modifications are transferable to other immunoglobulin heavy andlight chains, resulting in similar patterns of preferential pairing ofone immunoglobulin heavy chain with one of the two immunoglobulin lightchains in view of the following.

The VH:VL and CH1:CL interface residues in the interface betweenimmunoglobulin heavy and light chains are relatively well conserved(Padlan et al., 1986, Mol. Immunol. 23(9): 951-960). This sequenceconservation, a result of evolutionary constraints, increases thelikelihood that functionally active antibody binding domains will beformed during combinatorial pairing of light and heavy chains. As aresult of this sequence conservation, it follows that sequencemodifications in the specific examples noted above for D3H44, whichdrive preferential pairing, could transfer to other heavy and lightchain pair heterodimers with approximately equivalent results beingobtained with respect to preferential pairing, since this regiondisplays high sequence conservation across antibodies; Further, whensequence differences do occur, these usually lie distal to the CH1:CLinterface. This is particularly the case for the CH1 and CL domains.There is, however, some sequence variability at the antigen-binding sitewith respect to CDR (complementarity-determining regions) loop residues(and length), particularly for CDR-H3. Thus, in one embodiment, theheterodimer pairs according to the invention comprise heterodimers whereat least one heterodimer comprises one or more amino acid modificationsin the VH and/or VL domains that lie distal to the CDR loops when theamino acid sequence of the antigen-binding site is significantlydifferent from that of the D3H44 antibody. In another embodiment, theheterodimer pairs according to the invention comprise heterodimers whereat least one heterodimer comprises one or more amino acid modificationsin the VH and/or VL domains that lie proximal or distal to the CDRloops, when the amino acid sequence of the antigen-binding site issubstantially the same as that of the D3H44 antibody.

In one embodiment, the amino acid modifications described herein aretransferable to the immunoglobulin heavy and light chains of antibodiesbased on human or humanized IgG1/K. Non-limiting examples of such IgG1/κchains include Ofatumumab (for human) or Trastuzumab, Pertuzumab orBevacizumab (for humanized).

In another embodiment, the amino acid modifications described herein aretransferable to the immunoglobulin heavy and light chains of antibodiesutilizing commonly used VH and VL subgroups. Non-limiting examples ofsuch antibodies include Peruzumab.

In one embodiment, the amino acid modifications described herein aretransferable to the immunoglobulin heavy and light chains of antibodieshaving a framework close to germline. Examples of such antibodiesinclude Obinutuzumab.

In one embodiment, the amino acid modifications described herein aretransferable to the immunoglobulin heavy and light chains of antibodieshaving a VH:VL interdomain angle close to the average observed for heavyand light chain pairs. An example of this type of antibody includes, butis not limited to Pertuzumab. In another embodiment, the amino acidmodifications described herein are transferable to the immunoglobulinheavy and light chains of antibodies having canonical CL and CH1domains. Suitable examples of such antibodies include, but are notlimited to Trastuzumab.

In some embodiments, certain subsets of the amino acid modificationsdescribed herein are utilized in variant domains in antigen bindingconstructs provided above.

The Examples, Figs, and Tables demonstrate that amino acid modifications(e.g., within one or more Fab fragments comprising a variable region anda constant region) are transferable to other immunoglobulin heavy andlight chains, resulting in similar patterns of preferential pairing ofone immunoglobulin heavy chain with one of the two immunoglobulin lightchains.

Preferential Pairing

As described above, at least one heterodimer of the antigen bindingconstruct/heterodimer pairs according to the invention can comprise oneor more amino acid modifications to their immunoglobulin heavy and/orimmunoglobulin light chains such that the heavy chain of the oneheterodimer, for example H1, preferentially pairs with one of the lightchains, for example L1, rather than the other light chain, L2, and theheavy chain of the other heterodimer, H2, preferentially pairs with thelight chain, L2, rather than the light chain L1. In other words, thedesired, preferential pairing is considered to be between H1 and L1, andbetween H2 and L2. Preferential pairing between, for example, H1 and L1is considered to occur if the yield of the H1-L1 heterodimer is greaterthan the yield of the mispaired H1-L2 heterodimer when H1 is combinedwith L1 and L2, relative to the respective pairing of correspondingH1/L1 pair to H2/L2 pair without the one or more amino acidmodifications. Likewise, preferential pairing between H2 and L2 isconsidered to occur if the yield of the H2-L2 heterodimer is greaterthan the yield of the mispaired H2-L1 heterodimer when H2 is combinedwith L1 and L2, relative to the respective pairing of correspondingH1-L1 pair to H2-L2 pair without the one or more amino acidmodifications. In this context, an heterodimer comprising H1 and L1(H1-L1), or H2 and L2 (H2-L2), is referred to herein as a preferentiallypaired, correctly paired, obligate pair, or desired heterodimer, while aheterodimer comprising H1 and L2 (H1-L2), or H2 and L1 (H2-L1), isreferred to herein as a mispaired heterodimer. The set of mutationscorresponding to the two heavy chains and the two light chains meant toachieve selective pairing of H1-L1 and H2-L2 is referred to as a designset.

Thus, in one embodiment, when one immunoglobulin heavy chain of aheterodimer is co-expressed with two immunoglobulin light chains, therelative yield of the desired heterodimer is greater than 55%. Inanother embodiment, when one immunoglobulin heavy chain of a heterodimeris co-expressed with two immunoglobulin light chains, the relative yieldof the desired heterodimer is greater than 60%. In another embodiment,when one immunoglobulin heavy chain of a heterodimer is co-expressedwith two immunoglobulin light chains, the relative yield of the desiredheterodimer is greater than 70%. In another embodiment, when oneimmunoglobulin heavy chain of a heterodimer is co-expressed with twoimmunoglobulin light chains, the relative yield of the desiredheterodimer is greater than 80%. In another embodiment, when oneimmunoglobulin heavy chain of a heterodimer is co-expressed with twoimmunoglobulin light chains, the relative yield of the desiredheterodimer is greater than 90%. In another embodiment, when oneimmunoglobulin heavy chain of a heterodimer is co-expressed with twoimmunoglobulin light chains, the relative yield of the desiredheterodimer is greater than 95%.

In the above example, preferential pairing between H1-L1 is consideredto occur if the amount of the desired H1-L1 heterodimer is greater thanthe amount of the mispaired H1-L2 heterodimer when H1 is co-expressedwith L1 and L2. Similarly, preferential pairing between H2-L2 isconsidered to occur if the amount of the desired H2-L2 heterodimer isgreater than the amount of the mispaired H2-L2 heterodimer when H2 isco-expressed with L1 and L2. Thus, in one embodiment, when oneimmunoglobulin heavy chain of a heterodimer is co-expressed with twoimmunoglobulin light chains, the ratio of the desired heterodimer to themispaired heterodimer is greater than 1.25:1. In one embodiment, whenone immunoglobulin heavy chain of a heterodimer is co-expressed with twoimmunoglobulin light chains, the ratio of the desired heterodimer to themispaired heterodimer is greater than 1.5:1. In another embodiment, whenone immunoglobulin heavy chain of a heterodimer is co-expressed with twoimmunoglobulin light chains, the ratio of the desired heterodimer to themispaired heterodimer is greater than 2:1. In another embodiment, whenone immunoglobulin heavy chain of a heterodimer is co-expressed with twoimmunoglobulin light chains, the ratio of the desired heterodimer to themispaired heterodimer is greater than 3:1. In another embodiment, whenone immunoglobulin heavy chain of a heterodimer is co-expressed with twoimmunoglobulin light chains, the ratio of the desired heterodimer to themispaired heterodimer is greater than 5:1. In another embodiment, whenone immunoglobulin heavy chain of a heterodimer is co-expressed with twoimmunoglobulin light chains, the ratio of the desired heterodimer to themispaired heterodimer is greater than 10:1. In another embodiment, whenone immunoglobulin heavy chain of a heterodimer is co-expressed with twoimmunoglobulin light chains, the ratio of the desired heterodimer to themispaired heterodimer is greater than 25:1. In another embodiment, whenone immunoglobulin heavy chain of a heterodimer is co-expressed with twoimmunoglobulin light chains, the ratio of the desired heterodimer to themispaired heterodimer is greater than 50:1.

Thermal Stability of Heterodimers

In one embodiment, each heterodimer of the heterodimer pair according tothe invention has a thermal stability that is comparable to that of aheterodimer comprising the same immunoglobulin heavy and light chainsbut without the amino acid modifications to the CH1, CL, VH, or VLdomains described herein. In one embodiment, thermal stability isdetermined by measurement of melting temperature, or Tm. Thus, in oneembodiment, the thermal stability of a heterodimer according to theinvention is within about 10° C. of that of a heterodimer comprising thesame immunoglobulin heavy and light chains without the amino acidmodifications to the CH1, CL, VH, or VL domains described herein. Thus,in one embodiment, the thermal stability of a heterodimer according tothe invention is within about 5° C. of that of a heterodimer comprisingthe same immunoglobulin heavy and light chains without the amino acidmodifications to the CH1, CL, VH, or VL domains described herein. Inanother embodiment, the thermal stability of a heterodimer according tothe invention is within about 3° C. of that of a heterodimer comprisingthe same immunoglobulin heavy and light chains without the amino acidmodifications to the CH1, CL, VH, or VL domains described herein. Inanother embodiment, the thermal stability of a heterodimer according tothe invention is within about 2° C. of that of a heterodimer comprisingthe same immunoglobulin heavy and light chains without the amino acidmodifications to the CH1, CL, VH, or VL domains described herein. Inanother embodiment, the thermal stability of a heterodimer according tothe invention is within about 1.5° C. of that of a heterodimercomprising the same immunoglobulin heavy and light chains without theamino acid modifications to the CH1, CL, VH, or VL domains describedherein. In another embodiment, the thermal stability of a heterodimeraccording to the invention is within about 1° C. of that of aheterodimer comprising the same immunoglobulin heavy and light chainswithout the amino acid modifications to the CH1, CL, VH, or VL domainsdescribed herein. In another embodiment, the thermal stability of aheterodimer according to the invention is within about 0.5° C. of thatof a heterodimer comprising the same immunoglobulin heavy and lightchains without the amino acid modifications to the CH1, CL, VH, or VLdomains described herein. In another embodiment, the thermal stabilityof a heterodimer according to the invention is within about 0.25° C. ofthat of a heterodimer comprising the same immunoglobulin heavy and lightchains without the amino acid modifications to the CH1, CL, VH, or VLdomains described herein.

Affinity of Heterodimers for Antigen

In one embodiment, each heterodimer of the heterodimer pair has anaffinity for its respective antigen that is the same or comparable tothat of a heterodimer comprising the same immunoglobulin heavy and lightchains but without the amino acid modifications to the CH1, CL, VH, orVL domains described herein. In one embodiment, a heterodimer of theheterodimer pair has an affinity for its respective antigen that iswithin about 50 fold, or one order of magnitude, of that of aheterodimer comprising the same immunoglobulin heavy and light chainswithout the amino acid modifications to the CH1, CL, VH, or VL domainsdescribed herein. In one embodiment, a heterodimer of the heterodimerpair has an affinity for its respective antigen that is within about 25fold, or one order of magnitude, of that of a heterodimer comprising thesame immunoglobulin heavy and light chains without the amino acidmodifications to the CH1, CL, VH, or VL domains described herein. In oneembodiment, a heterodimer of the heterodimer pair has an affinity forits respective antigen that is within about 10 fold, or one order ofmagnitude, of that of a heterodimer comprising the same immunoglobulinheavy and light chains without the amino acid modifications to the CH1,CL, VH, or VL domains described herein. In another embodiment, aheterodimer of the heterodimer pair has an affinity for its respectiveantigen that is within about 5 fold of that of a heterodimer comprisingthe same immunoglobulin heavy and light chains without the amino acidmodifications to the CH1, CL, VH, or VL domains described herein. Inanother embodiment, a heterodimer of the heterodimer pair has anaffinity for its respective antigen that is within about 2.5 fold ofthat of a heterodimer comprising the same immunoglobulin heavy and lightchains without the amino acid modifications to the CH1, CL, VH, or VLdomains described herein. In another embodiment, a heterodimer of theheterodimer pair has an affinity for its respective antigen that iswithin about 2 fold of that of a heterodimer comprising the sameimmunoglobulin heavy and light chains without the amino acidmodifications to the CH1, CL, VH, or VL domains described herein. Inanother embodiment, a heterodimer of the heterodimer pair has anaffinity for its respective antigen that is within about 1.5 fold ofthat of a heterodimer comprising the same immunoglobulin heavy and lightchains without the amino acid modifications to the CH1, CL, VH, or VLdomains described herein. In another embodiment, a heterodimer of theheterodimer pair has an affinity for its respective antigen that iswithin about the same as that of a heterodimer comprising the sameimmunoglobulin heavy and light chains without the amino acidmodifications to the CH1, CL, VH, or VL domains described herein.

Additional Optional Modifications

In one embodiment, the immunoglobulin heavy and light chains of theheterodimer pairs according to the invention may be further modified(i.e., by the covalent attachment of various types of molecules) suchthat covalent attachment does not interfere with the preferentialpairing between heavy chain and light chains or affect the ability ofthe heterodimer to bind to its antigen, or affect its stability. Suchmodification include, for example, but not by way of limitation,glycosylation, acetylation, pegylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, linkage to a cellular ligand or other protein, etc. Any ofnumerous chemical modifications may be carried out by known techniques,including, but not limited to, specific chemical cleavage, acetylation,formylation, metabolic synthesis of tunicamycin, etc.

In another embodiment, the immunoglobulin heavy and light chains of theheterodimer pairs according to the invention may be conjugated (directlyor indirectly) to a therapeutic agent or drug moiety that modifies agiven biological response. Therapeutic agents or drug moieties are notto be construed as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein or polypeptide possessing adesired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, Onconase (or another cytotoxic RNase),Pseudomonas exotoxin, cholera toxin, or diphtheria toxin; a protein suchas tumor necrosis factor, alpha-interferon, beta-interferon, nervegrowth factor, platelet derived growth factor, tissue plasminogenactivator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I (see,International Publication No. WO 97/33899), AIM II (see, InternationalPublication No. WO 97/34911), Fas Ligand (Takahashi et al., 1994, J.Immunol., 6:1567), and VEGI (see, International Publication No. WO99/23105), a thrombotic agent or an anti-angiogenic agent, e.g.,angiostatin or endostatin; or, a biological response modifier such as,for example, a lymphokine (e.g., interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colonystimulating factor (“GM-CSF”), and granulocyte colony stimulating factor(“G-CSF”)), or a growth factor (e.g., growth hormone (“GH”)).

Moreover, in an alternate embodiment, an antibody can be conjugated totherapeutic moieties such as a radioactive materials or macrocyclicchelators useful for conjugating radiometal ions (see above for examplesof radioactive materials). In certain embodiments, the macrocyclicchelator is 1,4,7,10-tetraazacyclododecane-N,N′,N″,N″-tetraacetic acid(DOTA) which can be attached to the antibody via a linker molecule. Suchlinker molecules are commonly known in the art and described in Denardoet al., 1998, Clin Cancer Res. 4:2483; Peterson et al., 1999, Bioconjug.Chem. 10:553; and Zimmerman et al., 1999, Nucl. Med. Biol. 26:943.

In some embodiments, the immunoglobulin heavy and light chains of theheterodimer are expressed as fusion proteins comprising a tag tofacilitate purification and/or testing etc. As referred to herein, a“tag” is any added series of amino acids which are provided in a proteinat either the C-terminus, the N-terminus, or internally that contributesto the identification or purification of the protein. Suitable tagsinclude but are not limited to tags known to those skilled in the art tobe useful in purification and/or testing such as albumin binding domain(ABD), His tag, FLAG tag, glutathione-s-transferase, haemaglutinin (HA)and maltose binding protein. Such tagged proteins may also be engineeredto comprise a cleavage site, such as a thrombin, enterokinase or factorX cleavage site, for ease of removal, of the tag before, during or afterpurification.

In some embodiments, one or more of the cysteine residues at the bottomof the Fab domain in the light (position 214, Kabat numbering) and heavy(position 233, Kabat numbering) chain that form an interchain disulphidebond can be modified to serine or alanine or a non-cysteine or adistinct amino acid.

It is contemplated that additional amino acid modifications can be madeto the immunoglobulin heavy chains in order to increase the level ofpreferential pairing, and/or the thermal stability of the heterodimerpairs. For example, additional amino acid modifications can be made tothe immunoglobulin heavy chain Fc domain in order to drive preferentialpairing between heterodimer pairs relative to homodimer pairs. Suchamino acid modifications are known in the art and include, for example,those described, in US Patent Publication No. 2012/0149876.Alternatively, alternate strategies for driving preferential pairingbetween heterodimer pairs relative to homodimer pairs such as, forexample, “knobs into holes”, charged residues with ionic interactions,and strand-exchange engineered domain (SEED) technologies can also beemployed. The latter strategies have been described in the art and arereviewed in Klein et al, supra. Further discussion of Fc domains followsbelow.

Fc Domains

The constructs described herein can further include an Fc. In someaspects, the Fc comprises at least one or two CH3 domain sequences. Insome aspects, the Fc is coupled, with or without one or more linkers, toa first heterodimer and/or a second heterodimer. In some aspects, the Fcis a human Fc. In some aspects, the Fc is a human IgG or IgG1 Fc. Insome aspects, the Fc is a heterodimeric Fc. In some aspects, the Fccomprises at least one or two CH2 domain sequences.

In some aspects, the Fc comprises one or more modifications in at leastone of the CH3 domain sequences. In some aspects, the Fc comprises oneor more modifications in at least one of the CH2 domain sequences. Insome aspects, an Fc is a single polypeptide. In some aspects, an Fc ismultiple peptides, e.g., two polypeptides.

In some aspects, the Fc comprises one or more modifications in at leastone of the CH3 sequences. In some aspects, the Fc comprises one or moremodifications in at least one of the CH2 sequences. In some aspects, anFc is a single polypeptide. In some aspects, an Fc is multiple peptides,e.g., two polypeptides.

In some aspects, Fc is an Fc described in patent applicationsPCT/CA2011/001238, filed Nov. 4, 2011 or PCT/CA2012/050780, filed Nov.2, 2012, the entire disclosure of each of which is hereby incorporatedby reference in its entirety for all purposes.

In some aspects, a construct described herein comprises a heterodimericFc comprising a modified CH3 domain that has been asymmetricallymodified. The heterodimeric Fc can comprise two heavy chain constantdomain polypeptides: a first heavy chain polypeptide and a second heavychain polypeptide, which can be used interchangeably provided that Fccomprises one first heavy chain polypeptide and one second heavy chainpolypeptide. Generally, the first heavy chain polypeptide comprises afirst CH3 sequence and the second heavy chain polypeptide comprises asecond CH3 sequence.

Two CH3 sequences that comprise one or more amino acid modificationsintroduced in an asymmetric fashion generally results in a heterodimericFc, rather than a homodimer, when the two CH3 sequences dimerize. Asused herein, “asymmetric amino acid modifications” refers to anymodification where an amino acid at a specific position on a first CH3sequence is different from the amino acid on a second CH3 sequence atthe same position, and the first and second CH3 sequence preferentiallypair to form a heterodimer, rather than a homodimer. Thisheterodimerization can be a result of modification of only one of thetwo amino acids at the same respective amino acid position on eachsequence; or modification of both amino acids on each sequence at thesame respective position on each of the first and second CH3 sequences.The first and second CH3 sequence of a heterodimeric Fc can comprise oneor more than one asymmetric amino acid modification.

Table X provides the amino acid sequence of the human IgG Fc sequence,corresponding to amino acids 231 to 447 of the full-length human IgG1heavy chain. The CH3 sequence comprises amino acid 341-447 of thefull-length human IgG1 heavy chain.

Typically an Fc can include two contiguous heavy chain sequences (A andB) that are capable of dimerizing. In some aspects, one or bothsequences of an Fc include one or more mutations or modifications at thefollowing locations: L351, F405, Y407, T366, K392, T394, T350, S400,and/or N390, using EU numbering. In some aspects, an Fc includes amutant sequence shown in Table X. In some aspects, an Fc includes themutations of Variant 1 A-B. In some aspects, an Fc includes themutations of Variant 2 A-B. In some aspects, an Fc includes themutations of Variant 3 A-B. In some aspects, an Fc includes themutations of Variant 4 A-B. In some aspects, an Fc includes themutations of Variant 5 A-B.

TABLE X Human IgG1 APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS Fc sequenceHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS 231-447VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG (EU-QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIA numbering)VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 1) Variant IgG1 Fc sequence(231-447) Chain Mutations 1 A L351Y_F405A_Y407V 1 B T366L_K392M_T394W 2A L351Y_F405A_Y407V 2 B T366L_K392L_T394W 3 A T350V_L351Y_F405A_Y407V 3B T350V_T366L_K392L_T394W 4 A T350V_L351Y_F405A_Y407V 4 BT350V_T366L_K392M_T394W 5 A T350V_L351Y_S400E_F405A_Y407V 5 BT350V_T366L_N390R_K392M_T394W

The first and second CH3 sequences can comprise amino acid mutations asdescribed herein, with reference to amino acids 231 to 447 of thefull-length human IgG1 heavy chain. In one embodiment, the heterodimericFc comprises a modified CH3 domain with a first CH3 sequence havingamino acid modifications at positions F405 and Y407, and a second CH3sequence having amino acid modifications at position T394. In oneembodiment, the heterodimeric Fc comprises a modified CH3 domain with afirst CH3 sequence having one or more amino acid modifications selectedfrom L351Y, F405A, and Y407V, and the second CH3 sequence having one ormore amino acid modifications selected from T366L, T366I, K392L, K392M,and T394W.

In one embodiment, a heterodimeric Fc comprises a modified CH3 domainwith a first CH3 sequence having amino acid modifications at positionsL351, F405 and Y407, and a second CH3 sequence having amino acidmodifications at positions T366, K392, and T394, and one of the first orsecond CH3 sequences further comprising amino acid modifications atposition Q347, and the other CH3 sequence further comprising amino acidmodification at position K360. In another embodiment, a heterodimeric Fccomprises a modified CH3 domain with a first CH3 sequence having aminoacid modifications at positions L351, F405 and Y407, and a second CH3sequence having amino acid modifications at position T366, K392, andT394, one of the first or second CH3 sequences further comprising aminoacid modifications at position Q347, and the other CH3 sequence furthercomprising amino acid modification at position K360, and one or both ofsaid CH3 sequences further comprise the amino acid modification T350V.

In one embodiment, a heterodimeric Fc comprises a modified CH3 domainwith a first CH3 sequence having amino acid modifications at positionsL351, F405 and Y407, and a second CH3 sequence having amino acidmodifications at positions T366, K392, and T394 and one of said firstand second CH3 sequences further comprising amino acid modification ofD399R or D399K and the other CH3 sequence comprising one or more ofT411E, T411D, K409E, K409D, K392E and K392D. In another embodiment, aheterodimeric Fc comprises a modified CH3 domain with a first CH3sequence having amino acid modifications at positions L351, F405 andY407, and a second CH3 sequence having amino acid modifications atpositions T366, K392, and T394, one of said first and second CH3sequences further comprises amino acid modification of D399R or D399Kand the other CH3 sequence comprising one or more of T411E, T411D,K409E, K409D, K392E and K392D, and one or both of said CH3 sequencesfurther comprise the amino acid modification T350V.

In one embodiment, a heterodimeric Fc comprises a modified CH3 domainwith a first CH3 sequence having amino acid modifications at positionsL351, F405 and Y407, and a second CH3 sequence having amino acidmodifications at positions T366, K392, and T394, wherein one or both ofsaid CH3 sequences further comprise the amino acid modification ofT350V.

In one embodiment, a heterodimeric Fc comprises a modified CH3 domaincomprising the following amino acid modifications, where “A” representsthe amino acid modifications to the first CH3 sequence, and “B”represents the amino acid modifications to the second CH3 sequence:A:L351Y_F405A_Y407V, B:T366L_K392M_T394W, A:L351Y_F405A_Y407V,B:T366L_K392L_T394W, A:T350V_L351Y_F405A_Y407V,B:T350V_T366L_K392L_T394W, A:T350V_L351Y_F405A_Y407V,B:T350V_T366L_K392M_T394W, A:T350V_L351Y_S400E_F405A_Y407V, and/orB:T350V_T366L_N390R_K392M_T394W.

The one or more asymmetric amino acid modifications can promote theformation of a heterodimeric Fc in which the heterodimeric CH3 domainhas a stability that is comparable to a wild-type homodimeric CH3domain. In an embodiment, the one or more asymmetric amino acidmodifications promote the formation of a heterodimeric Fc domain inwhich the heterodimeric Fc domain has a stability that is comparable toa wild-type homodimeric Fc domain. In an embodiment, the one or moreasymmetric amino acid modifications promote the formation of aheterodimeric Fc domain in which the heterodimeric Fc domain has astability observed via the melting temperature (Tm) in a differentialscanning calorimetry study, and where the melting temperature is within4° C. of that observed for the corresponding symmetric wild-typehomodimeric Fc domain. In some aspects, the Fc comprises one or moremodifications in at least one of the CH3 sequences that promote theformation of a heterodimeric Fc with stability comparable to a wild-typehomodimeric Fc.

In one embodiment, the stability of the CH3 domain can be assessed bymeasuring the melting temperature of the CH3 domain, for example bydifferential scanning calorimetry (DSC). Thus, in a further embodiment,the CH3 domain has a melting temperature of about 68° C. or higher. Inanother embodiment, the CH3 domain has a melting temperature of about70° C. or higher. In another embodiment, the CH3 domain has a meltingtemperature of about 72° C. or higher. In another embodiment, the CH3domain has a melting temperature of about 73° C. or higher. In anotherembodiment, the CH3 domain has a melting temperature of about 75° C. orhigher. In another embodiment, the CH3 domain has a melting temperatureof about 78° C. or higher. In some aspects, the dimerized CH3 sequenceshave a melting temperature (Tm) of about 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 77.5, 78, 79, 80, 81, 82, 83, 84, or 85° C. or higher.

In some embodiments, a heterodimeric Fc comprising modified CH3sequences can be formed with a purity of at least about 75% as comparedto homodimeric Fc in the expressed product. In another embodiment, theheterodimeric Fc is formed with a purity greater than about 80%. Inanother embodiment, the heterodimeric Fc is formed with a purity greaterthan about 85%. In another embodiment, the heterodimeric Fc is formedwith a purity greater than about 90%. In another embodiment, theheterodimeric Fc is formed with a purity greater than about 95%. Inanother embodiment, the heterodimeric Fc is formed with a purity greaterthan about 97%. In some aspects, the Fc is a heterodimer formed with apurity greater than about 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% whenexpressed. In some aspects, the Fc is a heterodimer formed with a puritygreater than about 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% when expressed via asingle cell.

Additional methods for modifying monomeric Fc polypeptides to promoteheterodimeric Fc formation are described in International PatentPublication No. WO 96/027011 (knobs into holes), in Gunasekaran et al.(Gunasekaran K. et al. (2010) J Biol Chem. 285, 19637-46, electrostaticdesign to achieve selective heterodimerization), in Davis et al. (Davis,J H. et al. (2010) Prot Eng Des Sel; 23(4): 195-202, strand exchangeengineered domain (SEED) technology), and in Labrijn et al [Efficientgeneration of stable bispecific IgG1 by controlled Fab-arm exchange.Labrijn A F, Meesters J I, de Goeij B E, van den Bremer E T, Neijssen J,van Kampen M D, Strumane K, Verploegen S, Kundu A, Gramer M J, vanBerkel P H, van de Winkel J G, Schuurman J, Parren P W. Proc Natl AcadSci USA. 2013 Mar. 26; 110(13):5145-50.

In some embodiments an isolated construct described herein comprises anantigen binding construct which binds an antigen; and a dimeric Fcpolypeptide construct that has superior biophysical properties likestability and ease of manufacture relative to an antigen bindingconstruct which does not include the same Fc polypeptide. A number ofmutations in the heavy chain sequence of the Fc are known in the art forselectively altering the affinity of the antibody Fc for the differentFcgamma receptors. In some aspects, the Fc comprises one or moremodifications to promote selective binding of Fc-gamma receptors.

The CH2 domain is amino acid 231-340 of the sequence shown in Table X.Exemplary mutations are listed below:

S298A/E333A/K334A, S298/E333A/K334A/K326A (Lu Y, Vernes J M, Chiang N,et al. J Immunol Methods. 2011 Feb. 28; 365(1-2):132-141);

F243L/R292P/Y300L/V305I/P396L, F243L/R292P/Y30L/L235V/P396L (StavenhagenJ B, Gorlatov S, Tuaillon N, et al. Cancer Res. 2007 Sep. 15;67(18):8882-90; Nordstrom J L, Gorlatov S, Zhang W, et al Breast CancerRes. 2011 Nov. 30; 13(6):R1123);

F243L (Stewart R, Thom G, Levens M, et al. Protein Eng Des Set. 2011September; 24(9):671-8.), S298A/E333A/K334A (Shields R L, Namenuk A K,Hong K, et al. J Biol Chem. 2001 Mar. 2; 276(9):6591-604);

S239D/I332E/A330L, S239D/J32E (Lazar G A, Dang W, Karki S, et al. ProcNatl Acad Sci USA. 2006 Mar. 14; 103(11):4005-10);

S239D/S267E, S267E/L328F (Chu S Y, Vostiar I, Karki S, et al. MolImmunol. 2008 September; 45(15):3926-33);

S239D/D265S/S298A/I332E, S239E/S298A/K326A/A327H, G237F/S298A/A330L/I332E, S239D/1332E/S298A, S239D/K326E/A330L/I332E/S298A,G236A/S239D/D270L/I332E, S239E/S267E/1H268D, L234F/S267E/N325L,G237F/V266L/S267D and other mutations listed in WO2011/120134 andWO2011/120135, herein incorporated by reference. Therapeutic AntibodyEngineering (by William R. Strohl and Lila M. Strohl, WoodheadPublishing series in Biomedicine No 11, ISBN 1 907568 37 9, October2012) lists mutations on page 283.

In some embodiments a CH2 domain comprises one or more asymmetric aminoacid modifications. In some embodiments a CH2 domain comprises one ormore asymmetric amino acid modifications to promote selective binding ofa FcγR. In some embodiments the CH2 domain allows for separation andpurification of an isolated construct described herein.

FcRn Binding and PK Parameters

As is known in the art, binding to FcRn recycles endocytosed antibodyfrom the endosome back to the bloodstream (Raghavan et al., 1996, AnnuRev Cell Dev Biol 12:181-220; Ghetie et al., 2000, Annu Rev Immunol18:739-766). This process, coupled with preclusion of kidney filtrationdue to the large size of the full-length molecule, results in favorableantibody serum half-lives ranging from one to three weeks. Binding of Fcto FcRn also plays a key role in antibody transport. Thus, in oneembodiment, the constructs of the invention are able to bind FcRn.

Additional Modifications to Improve Effector Function.

In some embodiments a construct described herein can be modified toimprove its effector function. Such modifications are known in the artand include afucosylation, or engineering of the affinity of the Fcportion of antibodies towards an activating receptor, mainly FCGR3a forADCC, and towards C1q for CDC. The following Table Y summarizes variousdesigns reported in the literature for effector function engineering.

TABLE Y Reference Mutations Effect Lu, 2011, Ferrara AfucosylatedIncreased 2011, Mizushima 2011 ADCC Lu, 2011 S298A/E333A/K334A IncreasedADCC Lu, 2011 S298A/E333A/K334A/K326A Increased ADCC Stavenhagen, 2007F243L/R292P/Y300L/V305I/P396L Increased ADCC Nordstrom, 2011F243L/R292P/Y300L/L235V/P396L Increased ADCC Stewart, 2011 F243LIncreased ADCC Shields, 2001 S298A/E333A/K334A Increased ADCC Lazar,2006 S239D/I332E/A330L Increased ADCC Lazar, 2006 S239D/I332E IncreasedADCC Bowles, 2006 AME-D, not specified Increased mutations ADCC Heider,2011 37.1, mutations not Increased disclosed ADCC Moore, 2010S267E/H268F/S324T Increased CDC

Thus, in one embodiment, a construct described herein can include adimeric Fc that comprises one or more amino acid modifications as notedin the above table that confer improved effector function. In anotherembodiment, the construct can be afucosylated to improve effectorfunction.

Linkers

The constructs described herein can include one or more heterodimersdescribed herein operatively coupled to an Fc described herein. In someaspects, Fc is coupled to the one or more heterodimers with or withoutone or more linkers. In some aspects, Fc is directly coupled to the oneor more heterodimers. In some aspects, Fc is coupled to the one or moreheterodimers by one or more linkers. In some aspects, Fc is coupled tothe heavy chain of each heterodimer by a linker.

In some aspects, the one or more linkers are one or more polypeptidelinkers. In some aspects, the one or more linkers comprise one or moreantibody hinge regions. In some aspects, the one or more linkerscomprise one or more IgG1 hinge regions.

Methods of Preparing Heterodimer Pairs

As described above, the heterodimer pairs according to the invention cancomprise a first heterodimer and a second heterodimer, each heterodimercomprising an immunoglobulin heavy chain or fragment thereof having atleast a VH and CH1 domain, and an immunoglobulin light chain having a VLdomain and a CL domain. The immunoglobulin heavy chains andimmunoglobulin light chains of the heterodimer can readily be preparedusing recombinant DNA technology known in the art. Standard techniquessuch as, for example, those described in Sambrook and Russell, MolecularCloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 3rd ed., 2001); Sambrook et al., Molecular Cloning:A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 2nd ed., 1989); Short Protocols in Molecular Biology(Ausubel et al., John Wiley and Sons, New York, 4th ed., 1999); andGlick and Pasternak, Molecular Biotechnology: Principles andApplications of Recombinant DNA (ASM Press, Washington, D.C., 2nd ed.,1998) can be used for recombinant nucleic acid methods, nucleic acidsynthesis, cell culture, transgene incorporation, and recombinantprotein expression. Alternatively, the heterodimers and heterodimerpairs according to the invention can be chemically synthesized.

The nucleic acid and amino acid sequences of the immunoglobulin heavyand light chains of the antibodies from which the heterodimers arederived are either known in the art or can be readily determined usingnucleic acid and/or protein sequencing methods. Methods of geneticallyfusing the tags described herein to the immunoglobulin heavy and/orlight chains are known in the art, and some are described below and inthe Examples.

For example, methods of expressing and co-expressing immunoglobulinheavy and light chains in a host cell are well known in the art. Inaddition, methods of tagging heavy chains and/or light chains usingrecombinant DNA technology are also well known in the art. Expressionvectors and host cells suitable for expression of the heavy and lightchains are also well known in the art as described below.

Bispecific antibody production methods that do not rely on the use onlya single clonal or transient cell line expressing all four chains areknown in the art (Gramer, et al. (2013) mAbs 5, 962; Strop et al. (2012)J Mol Biol 420, 204.). These methods rely on a post production armexchange under redox conditions of the two pairs of light and heavychain involved in the formation of bispecific antibody (Redoxproduction). In this approach the H1:L1 and H2:L2 pairs can be expressedin two different cell lines to independently produce the two Fab arms.Subsequently, the two Fab arms are mixed under select redox conditionsto achieve re-association of the two unique heavy chain H1 and H2 toform the bispecific antibody comprising L1:H1:H2:L2 chains. One canenvision the use of the library/dataset of designs described herein inthe production of bispecific antibodies using the Redox productionmethod or modified versions of that method.

In certain embodiments, cell-free protein expression systems areutilized to co-express polypeptides (e.g., heavy and light chainpolypeptides) without the use of living cells. Instead, all componentsneeded to transcribe DNA to RNA and translate the RNA to protein (e.g.ribosomes, tRNAs, enzymes, cofactors, amino acids) are provided insolution for use in vitro. In certain embodiments, the in vitroexpression requires (1) the genetic template (mRNA or DNA) encoding theheavy and light chain polypeptides and (2) a reaction solutioncontaining the necessary transcriptional and translational molecularmachinery. In certain embodiments, cell extracts substantially supplycomponents of the reaction solution, for instance: RNA polymerases formRNA transcription, ribosomes for polypeptide translation, tRNA, aminoacids, enzymatic cofactors, an energy source, and cellular componentsessential for proper protein folding. Cell-free protein expressionsystems can be prepared using lysates derived from bacterial cells,yeast cells, insect cells, plant cells, mammalian cells, human cells orcombinations thereof. Such cell lysates can provide the correctcomposition and proportion of enzymes and building blocks required fortranslation. In some embodiments, cell membranes are removed to leaveonly the cytosolic and organelle components of the cell.

Several cell-free protein expression systems are known in the art asreviewed in Carlson et al. (2012) Biotechnol. Adv. 30:1185-1194. Forexample, cell-free protein expression systems are available based onprokaryotic or eukaryotic cells. Examples of prokaryotic cell-freeexpression systems include those from E. coli. Eukaryotic cell-freeprotein expression systems are available based on extracts from rabbitreticulocytes, wheat germ, and insect cells, for example. Suchprokaryotic and eukaryotic cell-free protein expression systems arecommercially available from companies such as Roche, Invitrogen, Qiagen,and Novagen. One skilled in the art would readily be able to selectsuitable cell-free protein expression systems that would producepolypeptides (e.g., heavy chain and light chain polypeptides) that arecapable of pairing with each other. Further, the cell-free proteinexpression system can also be supplemented with chaperones (e.g. BiP)and isomerases (e.g. disulphide isomerase) to improve the efficiency ofIgG folding.

In some embodiments, cell-free expression systems are utilized toco-express the heavy and light chain polypeptides from DNA templates(transcription and translation) or mRNA templates (translation only).

Vectors and Host Cells

Recombinant expression of heavy and light chains requires constructionof an expression vector containing a polynucleotide that encodes theheavy or light chain (e.g., antibody, or fusion protein). Once apolynucleotide encoding the heavy or light chain has been obtained, thevector for the production of the heavy or light chain may be produced byrecombinant DNA technology using techniques well known in the art. Thus,methods for preparing a protein by expressing a polynucleotidecontaining the heavy or light chain encoding nucleotide sequence aredescribed herein. Methods that are well known to those skilled in theart can be used to construct expression vectors containing heavy orlight chain coding sequences and appropriate transcriptional andtranslational control signals. These methods include, for example, invitro recombinant DNA techniques, synthetic techniques, and in vivogenetic recombination. The invention, thus, provides replicable vectorscomprising a nucleotide sequence encoding heavy or light chains,operably linked to a promoter.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce the modified heavy or light chains for use in themethod of the invention. In specific embodiments the heavy and lightchains for use in the method are co-expressed in the host cell forexpression of the entire immunoglobulin molecule, as detailed below.

A variety of host-expression vector systems may be utilized to expressthe modified heavy and light chains. Such host-expression systemsrepresent vehicles by which the coding sequences of interest may beproduced and subsequently purified, but also represent cells which may,when transformed or transfected with the appropriate nucleotide codingsequences, express the modified heavy and light chains in situ. Theseinclude but are not limited to microorganisms such as bacteria (e.g., E.coli and B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing the modifiedheavy and light chain coding sequences; yeast (e.g., SaccharomycesPichia) transformed with recombinant yeast expression vectors containingmodified heavy and light chain coding sequences; insect cell systemsinfected with recombinant virus expression vectors (e.g., baculovirus)containing modified heavy and light chain coding sequences; plant cellsystems infected with recombinant virus expression vectors (e.g.,cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing modified heavy and light chain coding sequences; ormammalian cell systems (e.g., COS, CHO, BHK, HEK-293, NSO, and 3T3cells) harboring recombinant expression constructs containing promotersderived from the genome of mammalian cells (e.g., metallothioneinpromoter) or from mammalian viruses (e.g., the adenovirus late promoter;the vaccinia virus 7.5K promoter). In certain embodiments, bacterialcells such as Escherichia coli, or eukaryotic cells, are used for theexpression of modified heavy and light chains, which is a recombinantantibody or fusion protein molecules. For example, mammalian cells suchas Chinese hamster ovary cells (CHO), in conjunction with a vector suchas the major intermediate early gene promoter element from humancytomegalovirus is an effective expression system for antibodies(Foecking et al., 1986, Gene 45:101; and Cockett et al., 1990,Bio/Technology 8:2). In a specific embodiment, the expression ofnucleotide sequences encoding the immunoglobulin heavy and light chainsof each heterodimer is regulated by a constitutive promoter, induciblepromoter or tissue specific promoter.

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the modified heavy and light chain coding sequences of interestmay be ligated to an adenovirus transcription/translation controlcomplex, e.g., the late promoter and tripartite leader sequence. Thischimeric gene may then be inserted in the adenovirus genome by in vitroor in vivo recombination. Insertion in a non-essential region of theviral genome (e.g., region E1 or E3) will result in a recombinant virusthat is viable and capable of expressing the modified heavy and lightchains in infected hosts (e.g., see Logan & Shenk, 1984, Proc. Natl.Acad. Sci. USA 81:355-359). Specific initiation signals may also berequired for efficient translation of inserted antibody codingsequences. These signals include the ATG initiation codon and adjacentsequences. Furthermore, the initiation codon must be in phase with thereading frame of the desired coding sequence to ensure translation ofthe entire insert. These exogenous translational control signals andinitiation codons can be of a variety of origins, both natural andsynthetic. The efficiency of expression may be enhanced by the inclusionof appropriate transcription enhancer elements, transcriptionterminators, etc. (see, e.g., Bittner et al., 1987, Methods in Enzymol.153:516-544).

The expression of the immunoglobulin heavy and light chains of theheterodimers may be controlled by any promoter or enhancer element knownin the art. Promoters which may be used to control the expression of thegene encoding modified heavy and light chains (e.g., antibody or fusionprotein) include, but are not limited to, the SV40 early promoter region(Bernoist and Chambon, 1981, Nature 290:304-310), the promoter containedin the 3′ long terminal repeat of Rous sarcoma virus (Yamamoto, et al.,1980, Cell 22:787-797), the herpes thymidine kinase promoter (Wagner etal., 1981, Proc. Natl. Acad. Sci. U.S.A. 78.1441-1445), the regulatorysequences of the metallothionein gene (Brinster et al., 1982, Nature296:39-42), the tetracycline (Tet) promoter (Gossen et al., 1995, Proc.Nat. Acad. Sci. USA 89:5547-5551); prokaryotic expression vectors suchas the β-lactamase promoter (Villa-Kamaroff et al, 1978, Proc. Natl.Acad. Sci. U.S.A. 75:3727-3731), or the tac promoter (DeBoer et al.,1983, Proc. Natl. Acad. Sci. U.S.A. 80:21-25; see also “Useful proteinsfrom recombinant bacteria” in Scientific American, 1980, 242:74-94);plant expression vectors comprising the nopaline synthetase promoterregion (Herrera-Estrella et al., Nature 303:209-213) or the cauliflowermosaic virus 35S RNA promoter (Gardner et al., 1981, Nucl. Acids Res.9:2871), and the promoter of the photosynthetic enzyme ribulosebiphosphate carboxylase (Herrera-Estrella et al., 1984, Nature310:115-120); promoter elements from yeast or other fungi such as theGal 4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK(phosphoglycerol kinase) promoter, alkaline phosphatase promoter, andthe following animal transcriptional control regions, which exhibittissue specificity and have been utilized in transgenic animals:elastase I gene control region which is active in pancreatic acinarcells (Swift et al., 1984, Cell 38:639-646; Ornitz et al., 1986, ColdSpring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology7:425-515); insulin gene control region which is active in pancreaticbeta cells (Hanahan, 1985, Nature 315:115-122), immunoglobulin genecontrol region which is active in lymphoid cells (Grosschedl et al.,1984, Cell 38:647-658; Adames et al., 1985, Nature 318:533-538;Alexander et al., 1987, Mol. Cell. Biol. 7:1436-1444), mouse mammarytumor virus control region which is active in testicular, breast,lymphoid and mast cells (Leder et al., 1986, Cell 45:485-495), albumingene control region which is active in liver (Pinkert et al., 1987,Genes and Devel. 1:268-276), alpha-fetoprotein gene control region whichis active in liver (Krumlauf et al., 1985, Mol. Cell. Biol. 5:1639-1648;Hammer et al., 1987, Science 235:53-58; alpha 1-antitrypsin gene controlregion which is active in the liver (Kelsey et al., 1987, Genes andDevel. 1:161-171), beta-globin gene control region which is active inmyeloid cells (Mogram et al., 1985, Nature 315:338-340; Kollias et al.,1986, Cell 46:89-94; myelin basic protein gene control region which isactive in oligodendrocyte cells in the brain (Readhead et al., 1987,Cell 48:703-712); myosin light chain-2 gene control region which isactive in skeletal muscle (Sani, 1985, Nature 314:283-286);neuronal-specific enolase (NSE) which is active in neuronal cells(Morelli et al., 1999, Gen. Virol. 80:571-83); brain-derivedneurotrophic factor (BDNF) gene control region which is active inneuronal cells (Tabuchi et al., 1998, Biochem. Biophysic. Res. Com.253:818-823); glial fibrillary acidic protein (GFAP) promoter which isactive in astrocytes (Gomes et al., 1999, Braz J Med Biol Res 32(5):619-631; Morelli et al., 1999, Gen. Virol. 80:571-83) and gonadotropicreleasing hormone gene control region which is active in thehypothalamus (Mason et al., 1986, Science 234:1372-1378).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Expression from certainpromoters can be elevated in the presence of certain inducers; thus,expression of the genetically engineered fusion protein may becontrolled. Furthermore, different host cells have characteristic andspecific mechanisms for the translational and post-translationalprocessing and modification (e.g., glycosylation, phosphorylation ofproteins). Appropriate cell lines or host systems can be chosen toensure the desired modification and processing of the foreign proteinexpressed. For example, expression in a bacterial system will produce anunglycosylated product and expression in yeast will produce aglycosylated product. Eukaryotic host cells that possess the cellularmachinery for proper processing of the primary transcript (e.g.,glycosylation, and phosphorylation) of the gene product may be used.Such mammalian host cells include, but are not limited to, CHO, VERY,BHK, Hela, COS, MDCK, HEK-293, 3T3, WI38, NSO, and in particular,neuronal cell lines such as, for example, SK-N-AS, SK-N-FI, SK-N-DZhuman neuroblastomas (Sugimoto et al., 1984, J. Natl. Cancer Inst. 73:51-57), SK-N-SH human neuroblastoma (Biochim. Biophys. Acta, 1982, 704:450-460), Daoy human cerebellar medulloblastoma (He et al., 1992, CancerRes. 52: 1144-1148) DBTRG-05MG glioblastoma cells (Kruse et al., 1992,In Vitro Cell. Dev. Biol. 28A: 609-614), IMR-32 human neuroblastoma(Cancer Res., 1970, 30: 2110-2118), 1321 N1 human astrocytoma (Proc.Natl. Acad. Sci. USA, 1977, 74: 4816), MOG-G-CCM human astrocytoma (Br.J. Cancer, 1984, 49: 269), U87MG human glioblastoma-astrocytoma (ActaPathol. Microbiol. Scand., 1968, 74: 465-486), A172 human glioblastoma(Olopade et al., 1992, Cancer Res. 52: 2523-2529), C6 rat glioma cells(Benda et al., 1968, Science 161: 370-371), Neuro-2a mouse neuroblastoma(Proc. Natl. Acad. Sci. USA, 1970, 65: 129-136), NB41A3 mouseneuroblastoma (Proc. Natl. Acad. Sci. USA, 1962, 48: 1184-1190), SCPsheep choroid plexus (Bolin et al., 1994, J. Virol. Methods 48:211-221), G355-5, PG-4 Cat normal astrocyte (Haapala et al., 1985, J.Virol. 53: 827-833), Mpf ferret brain (Trowbridge et al., 1982, In Vitro18: 952-960), and normal cell lines such as, for example, CTX TNA2 ratnormal cortex brain (Radany et al., 1992, Proc. Natl. Acad. Sci. USA 89:6467-6471) such as, for example, CRL7030 and Hs578Bst. Furthermore,different vector/host expression systems may effect processing reactionsto different extents.

For long-term, high-yield production of recombinant proteins, stableexpression is often preferred. For example, cell lines that stablyexpress the modified heavy and light chains of the invention (e.g.,antibody or fusion protein) may be engineered. Rather than usingexpression vectors that contain viral origins of replication, host cellscan be transformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched medium, and then areswitched to a selective medium. The selectable marker in the recombinantplasmid confers resistance to the selection and allows cells to stablyintegrate the plasmid into their chromosomes and grow to form foci thatin turn can be cloned and expanded into cell lines.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), and adeninephosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes can beemployed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection fordhfr, which confers resistance to methotrexate (Wigler et al., 1980,Natl. Acad. Sci. USA 77:3567; O'Hare et al., 1981, Proc. Natl. Acad.Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid(Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, whichconfers resistance to the aminoglycoside G-418 (Colberre-Garapin et al.,1981, J. Mol. Biol. 150:1); and hygro, which confers resistance tohygromycin (Santerre et al., 1984, Gene 30:147) genes.

Co-Expression of Heavy Chains and Light Chains

The immunoglobulin heavy chains and light chains of the heterodimerpairs according to the invention can be co-expressed in mammalian cells,as noted above. In one embodiment, one heavy chain is co-expressed withtwo different light chains in a LCCA design set as described above,where the heavy chain preferentially pairs with one of the two lightchains. In another embodiment, two heavy chains are co-expressed withtwo different light chains, where each heavy chain preferentially pairswith one of the light chains.

Testing of Heterodimer Pairs

As described above, at least one heterodimer of the heterodimer pairsaccording to the invention can comprise one or more amino acidmodifications to their immunoglobulin heavy and/or immunoglobulin lightchains such that when the two heavy chains and two light chains of theheterodimer pair are co-expressed in a mammalian cell, the heavy chainof the first heterodimer preferentially pairs with one of the lightchains rather than the other. Likewise, the heavy chain of the secondheterodimer preferentially pairs with the second light chain rather thanthe first. The degree of preferential pairing can be assessed, forexample, by using the methods described below. The affinity of eachheterodimer of the heterodimer pair for its respective antigen can betested as described below. The thermal stability of each heterodimer ofthe heterodimer pair can also be tested as described below.

Methods to Measure Preferential Pairing

LCCA

In one embodiment, preferential pairing between immunoglobulin heavy andlight chains is determined by performing a Light Chain Competition Assay(LCCA). Co-owned patent application PCT/US2013/063306, filed Oct. 3,2013, describes various embodiments of LCCA and is herein incorporatedby reference in its entirety for all purposes. The method allowsquantitative analysis of the pairing of heavy chains with specific lightchains within the mixture of co-expressed proteins and can be used todetermine if one particular immunoglobulin heavy chain selectivelyassociates with either one of two immunoglobulin light chains when theheavy chain and light chains are co-expressed. The method is brieflydescribed as follows: At least one heavy chain and two different lightchains are co-expressed in a cell, in ratios such that the heavy chainis the limiting pairing reactant; optionally separating the secretedproteins from the cell; separating the immunoglobulin light chainpolypeptides bound to heavy chain from the rest of the secreted proteinsto produce an isolated heavy chain paired fraction; detecting the amountof each different light chain in the isolated heavy chain fraction; andanalyzing the relative amount of each different light chain in theisolated heavy chain fraction to determine the ability of the at leastone heavy chain to selectively pair with one of the light chains.

The method provides reasonable throughput and is robust (i.e.insensitive to minor changes in operation, such as user or flow rate)and accurate. The method provides a sensitive assay that can measure theeffects of small variations in the protein sequences. Promiscuousprotein-protein; domain-domain; chain-chain interactions over largesurface areas usually require multiple mutations (swaps) in order tointroduce selectivity. The protein products do not need to be isolatedand purified which enables more efficient screening. Further detailsregarding an embodiment of this method are described in the Examples.

Alternative Methods to Determine Preferential Pairing

Alternative methods for detecting preferential pairing include usingLC-MS (Liquid chromatography-Mass spectrometry) to quantify the relativeheterodimer populations including each light chain using differences intheir molecular weight to identify each distinct species. An antigenactivity assay could also be used to quantify relative heterodimerpopulations containing each light chain whereby the degree of bindingmeasured (relative to controls) would be used to estimate eachrespective heterodimer population.

Additional methods such as SMCA are described in the Examples, Figs, andTables.

Thermal Stability

The thermal stability of the heterodimers can be determined according tomethods known in the art. The melting temperature of each heterodimer isindicative of its thermal stability. The melting point of theheterodimer may be measured using techniques such as differentialscanning calorimetry (Chen et al (2003) Pharm Res 20:1952-60; Ghirlandoet al (1999) Immunol Lett 68:47-52). Alternatively, the thermalstability of the heterodimer may be measured using circular dichroism(Murray et al. (2002) J. Chromatogr Sci 40:343-9).

Affinity for Antigen

The binding affinity of the heterodimers for their respective antigensand the off-rate of the interaction can be determined by competitivebinding assays according to methods well known in the art. One exampleof a competitive binding assay is a radioimmunoassay comprising theincubation of labeled antigen (e.g., 3H or 1251 with a molecule ofinterest (e.g., heterodimers of the present invention) in the presenceof increasing amounts of unlabeled antigen, and the detection of themolecule bound to the labeled ligand. The affinity of the heterodimer ofthe present invention for the antigen and the binding off-rates can bedetermined from the saturation data by Scatchard analysis.

The kinetic parameters of a heterodimer according to the invention mayalso be determined using surface plasmon resonance (SPR) based assaysknown in the art (e.g., BIAcore kinetic analysis). For a review ofSPR-based technology see Mullet et al., 2000, Methods 22: 77-91; Dong etal., 2002, Review in Mol. Biotech., 82: 303-23; Fivash et al., 1998,Current Opinion in Biotechnology 9: 97-101; Rich et al., 2000, CurrentOpinion in Biotechnology 11: 54-61. Additionally, any of the SPRinstruments and SPR based methods for measuring protein-proteininteractions described in U.S. Pat. Nos. 6,373,577; 6,289,286;5,322,798; 5,341,215; 6,268,125 are contemplated in the methods of theinvention. FACS can also be used to measured affinity, as is known inthe art.

Pharmaceutical Compositions

The present invention also provides pharmaceutical compositionscomprising the heterodimers or heterodimer pairs described herein. Suchcompositions comprise a therapeutically effective amount of theheterodimer or heterodimer pair, and a pharmaceutically acceptablecarrier. In a specific embodiment, the term “pharmaceuticallyacceptable” means approved by a regulatory agency of the Federal or astate government or listed in the U.S. Pharmacopeia or other generallyrecognized pharmacopeia for use in animals, and more particularly inhumans. The term “carrier” refers to a diluent, adjuvant, excipient, orvehicle with which the therapeutic is administered. Such pharmaceuticalcarriers can be sterile liquids, such as water and oils, including thoseof petroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Water is a preferredcarrier when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectable solutions. Suitable pharmaceutical excipients include starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene, glycol, water, ethanol and thelike. The composition, if desired, can also contain minor amounts ofwetting or emulsifying agents, or pH buffering agents. Thesecompositions can take the form of solutions, suspensions, emulsion,tablets, pills, capsules, powders, sustained-release formulations andthe like. The composition can be formulated as a suppository, withtraditional binders and carriers such as triglycerides. Oral formulationcan include standard carriers such as pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, etc. Examples of suitable pharmaceutical carriersare described in “Remington's Pharmaceutical Sciences” by E. W. Martin.Such compositions will contain a therapeutically effective amount of thecompound, preferably in purified form, together with a suitable amountof carrier so as to provide the form for proper administration to thepatient. The formulation should suit the mode of administration.

In certain embodiments, the composition comprising the heterodimer orheterodimer pair is formulated in accordance with routine procedures asa pharmaceutical composition adapted for intravenous administration tohuman beings. Typically, compositions for intravenous administration aresolutions in sterile isotonic aqueous buffer. Where necessary, thecomposition may also include a solubilizing agent and a local anestheticsuch as lignocaine to ease pain at the site of the injection. Generally,the ingredients are supplied either separately or mixed together in unitdosage form, for example, as a dry lyophilized powder or water freeconcentrate in a hermetically sealed container such as an ampoule orsachette indicating the quantity of active agent. Where the compositionis to be administered by infusion, it can be dispensed with an infusionbottle containing sterile pharmaceutical grade water or saline. Wherethe composition is administered by injection, an ampoule of sterilewater for injection or saline can be provided so that the ingredientsmay be mixed prior to administration.

In certain embodiments, the compositions described herein are formulatedas neutral or salt forms. Pharmaceutically acceptable salts includethose formed with anions such as those derived from hydrochloric,phosphoric, acetic, oxalic, tartaric acids, etc., and those formed withcations such as those derived from sodium, potassium, ammonium, calcium,ferric hydroxide isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

The amount of the composition described herein which will be effectivein the treatment, inhibition and prevention of a disease or disorderassociated with aberrant expression and/or activity of a therapeuticprotein can be determined by standard clinical techniques. In addition,in vitro assays may optionally be employed to help identify optimaldosage ranges. The precise dose to be employed in the formulation willalso depend on the route of administration, and the seriousness of thedisease or disorder, and should be decided according to the judgment ofthe practitioner and each patient's circumstances. Effective doses areextrapolated from dose-response curves derived from in vitro or animalmodel test systems.

Uses of Heterodimer Pairs

As described above, the heterodimer pairs according to the invention cancomprise a first heterodimer and a second heterodimer, where theimmunoglobulin heavy chain and the immunoglobulin light chain of eachheterodimer is derived or engineered from a known therapeutic antibodyor from a known antibody that binds a molecule. Thus, it is contemplatedthat heterodimers derived or engineered from these antibodies could beused for the treatment or prevention of the same disease, disorder, orinfection that the known therapeutic antibody or known antibody can beused for.

Thus, in one embodiment, heterodimer pairs according to the inventionthat comprise a heterodimer with heavy and light chains derived from atherapeutic antibody that can be used for the treatment and/orprevention of cancer and related disorders, can also be used for thetreatment and/or prevention of cancer and related disorders.

In another embodiment, heterodimer pairs according to the invention thatcomprise a heterodimer with heavy and light chains derived from atherapeutic antibody that can be used for preventing, treating, ormanaging the symptoms of an inflammatory disorder in a subject, can alsobe used for preventing, treating, or managing the symptoms of aninflammatory disorder in a subject.

In another embodiment, heterodimer pairs according to the invention thatcomprise a heterodimer with heavy and light chains derived from atherapeutic antibody that can be used for the treatment or prevention ofautoimmune disease or inflammatory disease in a subject, can also beused for the treatment or prevention of autoimmune disease orinflammatory disease in a subject.

In another embodiment, heterodimer pairs according to the invention thatcomprise a heterodimer with heavy and light chains derived from atherapeutic antibody that can be used for the treatment or prevention ofan infectious disease in a subject, can also be used for the treatmentor prevention of an infectious disease in a subject.

In another embodiment, heterodimer pairs according to the invention thatcomprise a heterodimer with heavy and light chains derived from atherapeutic antibody that can be used for the treatment of vasculardisease in a subject, can also be used for the treatment of vasculardisease in a subject.

In another embodiment, the heterodimer pairs according to the inventionmay also be advantageously utilized in combination with othertherapeutic agents known in the art for the treatment or prevention of acancer, autoimmune disease, inflammatory disorders or infectiousdiseases. In a specific embodiment, the heterodimer pairs according tothe invention may be used in combination with monoclonal or chimericantibodies, lymphokines, or hematopoietic growth factors (such as, e.g.,IL-2, IL-3 and IL-7), which, for example, serve to increase the numberor activity of effector cells which interact with the molecules and,increase immune response. The heterodimer pairs according to theinvention may also be advantageously utilized in combination with one ormore drugs used to treat a disease, disorder, or infection such as, forexample anti-cancer agents, anti-inflammatory agents or anti-viralagents.

Kits

The present invention additionally provides for kits comprising one ormore heterodimer pairs. Individual components of the kit would bepackaged in separate containers and, associated with such containers,can be a notice in the form prescribed by a governmental agencyregulating the manufacture, use or sale of pharmaceuticals or biologicalproducts, which notice reflects approval by the agency of manufacture,use or sale. The kit may optionally contain instructions or directionsoutlining the method of use or administration regimen for theheterodimer pairs.

When one or more components of the kit are provided as solutions, forexample an aqueous solution, or a sterile aqueous solution, thecontainer means may itself be an inhalant, syringe, pipette, eyedropper, or other such like apparatus, from which the solution may beadministered to a subject or applied to and mixed with the othercomponents of the kit.

The components of the kit may also be provided in dried or lyophilizedform and the kit can additionally contain a suitable solvent forreconstitution of the lyophilized components. Irrespective of the numberor type of containers, the kits of the invention also may comprise aninstrument for assisting with the administration of the composition to apatient. Such an instrument may be an inhalant, nasal spray device,syringe, pipette, forceps, measured spoon, eye dropper or similarmedically approved delivery vehicle.

Computer Implementation

In one embodiment, a computer comprises at least one processor coupledto a chipset. Also coupled to the chipset are a memory, a storagedevice, a keyboard, a graphics adapter, a pointing device, and a networkadapter. A display is coupled to the graphics adapter. In oneembodiment, the functionality of the chipset is provided by a memorycontroller hub and an I/O controller hub. In another embodiment, thememory is coupled directly to the processor instead of the chipset.

The storage device is any device capable of holding data, like a harddrive, compact disk read-only memory (CD-ROM), DVD, or a solid-statememory device. The memory holds instructions and data used by theprocessor. The pointing device may be a mouse, track ball, or other typeof pointing device, and is used in combination with the keyboard toinput data into the computer system. The graphics adapter displaysimages and other information on the display. The network adapter couplesthe computer system to a local or wide area network.

As is known in the art, a computer can have different and/or othercomponents than those described previously. In addition, the computercan lack certain components. Moreover, the storage device can be localand/or remote from the computer (such as embodied within a storage areanetwork (SAN)).

As is known in the art, the computer is adapted to execute computerprogram modules for providing functionality described herein. As usedherein, the term “module” refers to computer program logic utilized toprovide the specified functionality. Thus, a module can be implementedin hardware, firmware, and/or software. In one embodiment, programmodules are stored on the storage device, loaded into the memory, andexecuted by the processor.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims.

EXAMPLES

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of protein chemistry, biochemistry,recombinant DNA techniques and pharmacology, within the skill of theart. Such techniques are explained fully in the literature. See, e.g.,T. E. Creighton, Proteins: Structures and Molecular Properties (W.H.Freeman and Company, 1993); A. L. Lehninger, Biochemistry (WorthPublishers, Inc., current addition); Sambrook, et al., MolecularCloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology(S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington'sPharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack PublishingCompany, 1990); Carey and Sundberg Advanced Organic Chemistry 3rd Ed.(Plenum Press) Vols A and B(1992).

Example 1: Preparation of Constructs Encoding D3H44 IgG Heavy Chains andD3H44 IgG Light Chains

The wild-type heavy and light chains of the anti-tissue factor antibodyD3H44 for use in the co-expression sets described herein were preparedas follows. D3H44 Fab light (AJ308087.1) and heavy (AJ308086.1) chainsequences were taken from GenBank (Tables A, A1, and A2), genesynthesized and codon optimized for mammalian expression. Light chainvector inserts, consisting of a 5′-EcoRI cutsite-HLA-A signal peptide-HAor FLAG tag-Light chain Ig clone-‘TGA stop’-BamH1 cutsite-3′, wereligated into a pTT5 vector (Durocher Y et al., Nucl. Acids Res. 2002;30, No. 2 e9). The resulting vector+insert were sequenced to confirmcorrect reading frame and sequence of the coding DNA. Likewise, heavychain vector inserts, consisting of a 5′-EcoR1 cutsite-HLA-A signalpeptide-heavy chain clone (terminating at T238; see TableA1)-ABD2-His6tag-TGA stop-BamH1 cutsite-3′ (“His6” disclosed as SEQ IDNO: 2), were ligated into a pTT5 vector (ABD; albumin binding domain).The resulting vector+insert were also sequenced to confirm correctreading frame and sequence of the coding DNA. The various D3H44constructs were generated either by gene synthesis or by site-directedmutagenesis (Braman J, Papworth C & Greener A., Methods Mol. Biol.(1996) 57:31-44).

Heavy and light chains were tagged at the C- and N-terminalsrespectively, in order to facilitate the assessment of preferentialpairing via a competition assay-SPR screen. The ABD2-His6 heavy chaintag (“His6” disclosed as SEQ ID NO: 2) specifically allowed HC-LCcomplexes to be captured on an anti-his tag SPR chip surface, whilstFLAG and HA light chain tags allowed the relative LC1 and LC2populations to be quantified.

Example 2: Assessment of Preferential Pairing of Heterodimers inCo-Expression Sets Comprising Variable Domain Modifications in D3H44 IgGLight and/or Heavy Chains

The ability of heterodimers to preferentially pair in co-expression setscomprising D3H44 heavy and light chains with modified V_(L) and/or V_(H)domains was determined and the results are shown in FIGS. 1A-1B. Theresults provided in FIGS. 1A-1B are preliminary and a more complete setof results is provided below. The amino acid modifications shown inFIGS. 1A-3 are identified with reference to the amino acid sequence ofD3H44 heavy chain and D3H44 light chain. See Tables A, A1, and A2.

One D3H44 heavy chain construct was co-expressed with two unique D3H44light chain constructs and the relative light chain pairing specificity(e.g. H1-L1:H1-L2) was determined from a competition assay-SPR screen(Column entitled “Competition assay screen results” in FIGS. 1A-1).Selected heterodimer hits were verified via a light chain competitionassay verification whereby L1:L2 DNA ratios were varied by 40:60, 50:50and 60:40 during transfection (Column entitled “Competition assayverification results” in FIGS. 1A-1). Heavy chain were kept in limitingquantities (i.e. HC<L1+L2) for both competition assay screens andverifications. A schematic representing the design of the assay is shownin FIG. 8.

The methods were carried out as follows: The Light Chain CompetitionAssay (LCCA) quantifies the relative pairing of one heavy chain for atleast two unique light chains. The assay and the preceding steps can besummarized as follows: 1. Concomitant expression of heavy and lightchains, with the heavy chain being in limiting amounts (e.g.HC:LC1:LC2=1:1:1), 2. Isolation of HC-LC complexes—achieved by bindingheavy chains to the SPR chip via a his-tag pull-down, and 3)Quantification of relative HC-LC populations (i.e. H1-L1:H1-L2). In theSPR format, antibodies specific for unique light chain-taggedpopulations are used for the quantification. Note: This assay can becarried out with or without the H-L disulphide. A schematic diagramrepresenting the method is shown in FIG. 9.

Transfection Method

Co-expression sets comprising one heavy chain and two light chainconstructs prepared as described in Example 1 were transfected intoCHO-3E7 cells as follows. CHO-3E7 cells, at a density of 1.7-2×10⁶cells/ml, were cultured at 37° C. in FREESTYLE™ F17 medium (Invitrogencat #A-1383501) supplemented with 4 mM glutamine and 0.1% Pluronic F-68(Invitrogen cat #24040-032). A total volume of 2 ml were transfectedwith a total of 2 ug DNA using PEIPRO® (Polyplus cat #115-010) at aDNA:PEI ratio of 1:2.5. Twenty-four hours after the addition of theDNA-PEI mixture, the cells were transferred to 32° C. Supernatants weretested for expression on day 7 by non-reducing SDS-PAGE analysisfollowed by Coommassie blue staining to visualize the bands. HC:LCratios are as indicated in Table 7.

TABLE 7 DNA quantity used for transfection (ng) HC:L1:L2Stuffer{circumflex over ( )} ratio Experiment HC LC1 LC2 DNA 1:1:1Competition 333 333 333 1000 assay screen 1:1:1 Competition 333 333 3331000 assay verification 1:0.8:1.2 Competition 333 266 400 1000 assayverification 1:1:1 Competition 333 333 333 1000 assay verification1:1.2:0.8 Competition 333 400 266 1000 assay verification {circumflexover ( )}Stuffer DNA: _(P)TT5 vector without a DNA insert.

Competition Assay SPR Method

The degree of preferential D3H44 light chain pairing to D3H44 heavychain in co-expression sets was assessed using an SPR-based readout ofunique epitope tags located at the N-terminus of each light chain.

Surface Plasmon resonance (SPR) supplies. GLM sensorchips, the BioradPROTEON™ amine coupling kit (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysulfosuccinimide (sNHS) andethanolamine), and 10 mM sodium acetate buffers were purchased fromBio-Rad Laboratories (Canada) Ltd. (Mississauga, ON). Recombinant Her-2protein was purchased from eBioscience (San Diego, Calif.).4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer,ethylenediaminetetraacetic acid (EDTA), and NaCl were purchased fromSigma-Aldrich (Oakville, ON). 10% Tween 20 solution was purchased fromTeknova (Hollister, Calif.).

SPR biosensor assays. All surface plasmon resonance assays were carriedout using a BioRad PROTEON™ XPR36 instrument (Bio-Rad Laboratories(Canada) Ltd. (Mississauga, ON)) with PBST running buffer (PBS TeknovaInc with 0.05% Tween20) at a temperature of 25° C. The anti-penta His(SEQ ID NO: 3) capture surface was generated using a GLM sensorchipactivated by a 1:5 dilution of the standard BioRad sNHS/EDC solutionsinjected for 140 s at 100 μL/min in the analyte (horizontal) direction.Immediately after the activation, a 25 μg/mL solution of anti-penta Hisantibody (SEQ ID NO: 3) (Qiagen Inc.) in 10 mM NaOAc pH 4.5 was injectedin the analyte (vertical) direction at a flow rate of 25 μL/min untilapproximately 3000 resonance units (RUs) were immobilized. Remainingactive groups were quenched by a 140 s injection of 1M ethanolamine at100 μL/min in the analyte direction, and this also ensuresmock-activated interspots are created for blank referencing.

The screening of the heterodimers for binding to the anti-FLAG (SigmaInc.) and anti-HA (Roche Inc.) monoclonal antibodies occurred in twosteps: an indirect capture of the heterodimers onto the anti-penta Hissurface (SEQ ID NO: 3) in the ligand direction followed by an anti-FLAGand anti-HA injection in the analyte direction. First, one bufferinjection for 30 s at 100 uL/min in the ligand direction was used tostabilize the baseline. For each heterodimer capture, unpurifiedheterodimers in cell-culture media were diluted to 4% in PBST. One tofive heterodimers or controls (i.e. controls containing either 100%HA-light chain or 100% FLAG-light chain) were simultaneously injected inindividual ligand channels for 240 s at flow 25 L/min. This resulted ina saturating heterodimer capture of approximately 300 to 400 RUs ontothe anti-penta His surface (SEQ ID NO: 3). The first ligand channel wasleft empty to use as a blank control if required. This heterodimercapture step was immediately followed by two buffer injections in theanalyte direction to stabilize the baseline, and then 5 nM anti-FLAG and5 nM anti-HA were each injected in duplicate at 50 μL/min for 120 s witha 180 s dissociation phase, resulting in a set of binding sensorgramswith a buffer reference for each of the captured heterodimer. Wherepossible, the antigen to which the heterodimer binds can also beinjected over the last remaining analyte channel as an activity control.The heterodimers were regenerated by an 18 s pulse of 0.85% phosphoricacid for 18 s at 100 μL/min to prepare the anti-penta His surface (SEQID NO: 3) for the next injection cycle. Sensorgrams were aligned anddouble-referenced using the buffer blank injection and interspots, andthe resulting sensorgrams were analyzed using PROTEON™ Manager softwarev3.0.

The total percentage of L1 and L2 should, theoretically, add up to 100%.In practice, it was observed for some variants that the total amount ofL1 and L2 added up to significantly less than 100%. This discrepancy intotal light chain percentage is believed to be due in part to theoccurrence of variable non-specific binding during initial heterodimercapture on the SPR chip.

Example 3: Assessment of Preferential Pairing of Heterodimers inCo-Expression Sets Comprising Constant (C_(L) or C_(H1)) DomainModifications in D3H44 IgG Light and/or Heavy Chains

The ability of heterodimers to preferentially pair in co-expression setscomprising D3H44 heavy and light chains with modified C_(L) and/orC_(H1) domains was determined as described for heterodimers withvariable domain modifications in Example 2, and the results are shown inFIGS. 2A-2B. One D3H44 heavy chain construct was co-expressed with twounique D3H44 light chain constructs and the relative light chain pairingspecificity (e.g. H1-L1:H1-L2) was determined from a competitionassay-SPR screen (Column entitled “Competition assay screen results” inFIGS. 2A-2B). Selected heterodimer hits were confirmed via a modifiedcompetition assay verification where DNA ratios of L1:L2 were varied by40:60, 50:50 and 60:40 during transfection (Column entitled “Competitionassay verification results” in FIGS. 2A-2B). As described in Example 2,heavy chain was kept in limiting quantities (i.e. HC<L1+L2) for bothcompetition assay screens and verifications. Assessment of preferentialpairing was carried out as described in Example 2.

Example 4: Scale Up for Biophysical Characterization

Selected heterodimers, both paired and mispaired, were scaled up(typically to 50 ml) and purified as follows in order to test forthermal stability and antigen binding. Heterodimers HD100-HD 115, asshown in FIG. 3 were expressed and purified. The heavy and light chainof each heterodimer was expressed in 50 ml cultures of CHO-3E7 cellsunder the culture conditions described above. Cells were centrifuged andheterodimers purified by loading the supernatant on Fractogel columncharged with Nickel as described below.

Purification on Fractogel Column Charged with Nickel (his)

Charging the column with Nickel: Sequentially wash with 5 column volumes(CV) of 0.5 M NaCl (no pH adjustment), followed by 4 CV 200 mM of NiCl₂(Nickel) and 2 CV of 0.5 M NaCl pH 5.0. Sample loading and elution:Equilibrate column with 10 CV PBS. Load sample and wash with 10 CV ofwash buffer #1 (50 mM sodium phosphate pH 7.0, 300 mM NaCl) followed by10 CV of wash buffer #2 (50 mM sodium phosphate pH 7.0, 300 mM NaCl, 25mM Imidazole) to remove impurities bound to the column. The heterodimerswere eluted in fractions with wash buffer #1+300 mM Imidazole. Theprotein content of each fraction was tested by Bradford protein assay.Fractions containing protein were pooled. The purified heterodimers werethen assayed for antigen binding and thermal stability as described inExample 5.

Example 5: Thermal Stability and Antigen Affinity Measurements ofHeterodimers

The thermal stability and antigen affinity of selected heterodimer pairswas measured in order to compare these features with that of wild type,unmodified heavy chain-light chain pair. Correctly paired and mispairedheterodimers from co-expression sets were individually scaled up,purified (i.e. His tag affinity purification) and assessed for thermalstability and antigen binding as described below. The results are shownin FIG. 3.

Measurement of Thermal Stability

The thermal stability of selected heterodimer pairs was measured usingdifferential scanning calorimetry (DSC) as follows.

Each heterodimer was purified as described in Example 3 and diluted to0.2 mg/mL in PBS, and a total of 400 μL was used for DSC analysis with aVP-Capillary DSC (GE Healthcare). At the start of each DSC run, 5 bufferblank injections were performed to stabilize the baseline, and a bufferinjection was placed before each heterodimer injection for referencing.Each sample was scanned from 20 to 100° C. at a 60° C./hr rate, with lowfeedback, 8 sec filter, 5 min preTstat, and 70 psi nitrogen pressure.The resulting thermograms were referenced and analyzed using Origin 7software.

Thermal unfolding curves for the heterodimers tested are shown in FIG.4. The results indicate that the correctly paired heterodimer (from adesign perspective) is usually significantly more stable than theintended mispaired heterodimer (e.g. HD107 versus HD108). In addition,many of the correctly paired heterodimer exhibit a thermal stabilityclose to wild-type Fab (e.g. HD114).

Measurement of Antigen Affinity

The affinity of the heterodimer pairs for antigen (tissue factorextracellular domains) was measured using surface plasmon resonance(SPR) assays. All surface plasmon resonance assays were carried outusing a BioRad PROTEON™ XPR36 instrument (Bio-Rad Laboratories (Canada)Ltd. (Mississauga, ON) with PBST running buffer (PBS Teknova Inc with0.05% Tween20) at a temperature of 25° C. A purified tissue factor (TF)surface was generated using a GLM sensorchip activated by a 1:10dilution of the standard BioRad sNHS/EDC solutions injected for 140 s at100 μL/min in the ligand (vertical) direction. Immediately after theactivation, a 25 μg/mL solution of TF in 10 mM NaOAc pH 4.5 was injectedin the ligand direction at a flow rate of 25 μL/min until approximately1000 resonance units (RUs) were immobilized (or enough for a 100 RUmaximum response when flowing 60 nM FAB). Remaining active groups werequenched by a 140 s injection of 1M ethanolamine at 100 μL/min in theanalyte direction. For each injection series, two buffer blankinjections in the horizontal injection preceded the purifiedheterodimer. A 3-fold dilution series of each heterodimer (60 nM, 20 nM,6.7 nM, 2.2 nM) with a blank buffer control was simultaneously injectedat 50 μL/min for 120 s with a 20 minute dissociation, resulting in a setof binding sensorgrams with a buffer reference for each of theheterodimers. The heterodimer:TF complexes on the SPR surface wereregenerated by an 18 s pulse of 0.85% phosphoric acid for 18 s at 100μL/min to prepare the TF surface for the next injection cycle.Sensorgrams were aligned and double-referenced using the buffer blankinjection and interspots, and the resulting sensorgrams were analyzedusing a 1:1 binding model within the PROTEON™ Manager software v3.0.

The results indicate that the correctly paired heterodimer (from adesign perspective) exhibits a range of affinities for antigen, withsome designs showing wild-type like binding affinity for antigen (e.g.HD107 and HD 114).

Example 6: Size Exclusion Chromatography (SEC) Profiles of Wild-TypeTagged D3H44 Heterodimers and a Representative Sample of IndividualPreferentially Paired Heterodimers

Wild-type D3H44 heterodimer (one heavy chain and one light chain) with aC-terminus ABD2-His6 tag (“His6” disclosed as SEQ ID NO: 2) on the heavychain and an N-terminus FLAG tag on the light chain were expressed andpurified according to methods known in the art and similar to thosedescribed in Examples 1 and 4. Preferentially or correctly pairedheterodimers from co-expression sets (heterodimers HD100, HD105, andHD107, shown in FIG. 3) were individually scaled up and purified via Histag affinity purification as described in Example 4 and SEC.

SEC was carried out as follows. Heterodimer samples were separated usinga Superdex 200 HR 10/30 Pharmacia (GE Healthcare) column mounted on aPharmacia (GE Healthcare) ÄKTA Purifier system. Heterodimer samples(0.3-0.5 ml) in PBS were manually loaded into a 0.5 ml loop filled withPBS. Samples were than automatically injected onto the column andresolved at 0.5 ml/min with a 1 CV elution volume. Protein elution wasmonitored at OD₂₈₀ and collected in 1 ml fractions.

As shown in FIGS. 5A-5D, correctly paired heterodimers displayed SECprofiles close to that observed for wild-type heterodimer without aminoacid modifications (Main peak [*]:heterodimer). Equivalent results areobtained when the light chain of the wild-type heterodimer has aN-terminus HA tag.

Example 7: Additional Data Relating to the Stability of Heterodimers

Designs shown in Table 8 were highlighted as combinations of designdrivers with improved HC-LC selectivities.

TABLE 8 Design Set # Set # H1_mutation L1_mutation H2_mutationL2_mutation 11 C525 C526 S188L_V190Y V133S F174V_P175S_(—) S176L S188G12 V042 V043 V37E_F100D L89R_F98W WT WT 13 C532 C533 L143A_D144G_(—)Q124E_V133W_(—) K145T_Q179D_(—) V133A_Q160K_(—) Q179R Q160E_T180E S188FT178R 14 D146G_S186R Q124E_Q160E_(—) K145E_D146G_(—) Q124R_Q160K_(—)T178D Q179D_S188L T178R 15 C530 C531 D146G_Q179R Q124E_Q160E_(—)K145T_Q179D_(—) Q160K_T178R T178D S188L

Residue numbering follows Kabat nomenclature (Kabat E. A. et al., (1983)Sequence of Proteins of Immunological Interest National Institutes ofHealth, Bethesda).

The majority of the designs retained wild-type like thermal stability(Tm) and TF binding affinity as shown in Table 9.

TABLE 9 Tm (° C.) TF Binding KD (nM) H1 Readout H2 Readout Design H1-L1Tm H2-L2 Tm H1-L1 H2-L2 H1-L1 H1-L2 H2-L2 H2-L1 11 74.7 73.3 0.043 0.04083 24 98 1 12 76.2 76.0 0.052 99 10 88 15 13 67.5 71.5 0.087 0.086 104 187 14 14 84 1 74 2 15 70.1 76.3 0.082 0.071 93 1 83 24

Design 12 contains a wild-type H2-L2 pairing (see Table 8). As a result,H2-L2 Fab retains wild-type binding affinity. Wild-type anti-TF D3H44Fab Tm=˜76° C. (data not shown).

Example 8: Additional Heterodimers and Testing of Same

Additional heterodimer pairs as described in Table 10 were prepared andtested. These heterodimers were designed to increase Fab hotspotcoverage.

TABLE 10 Design H1_mutation* L1_mutation 26 A141G_V185A L135W 27A141I_K147T_D148G_Q175E_S183G_V185S F116A_V133G_S176F_T178A 28A141V_K147L_Q175E_S183G_V185S F116A_S131K_V133G_S176F_T178A 29L145K_D148G Q124E_V133D 30 D148G_Q175K Q124E_Q160E_T180E 31Q39D_A141G_V185A Q38R_L135W 32 Q39E Q38R Design H2_mutation L2_mutation26 A141W_K147Y_Q175E F116A_S131K_L135A 27 S181K_S183H_V185GF118W_Q124E_V133S_S176A_T178S_T180E 28 A141W_S181K_S183AF118W_V133S_S176A_T180E 29 L145E_K147T Q124R 30 L145E_K147T Q160K_T178R31 Q39R_A141W Q38D_F116A_L135A 32 Q39R Q38E_F98W *Residue numbering inTable 10 follows the convention used for residues in the crystalstructure of D3H44 Fab (PDB ID = 1JPT [Faelber K et al., J. Mol. Biol.(2001) 313: 83-97];www.resb.org/pdb/explore/explore.do?structureId=1JPT).

The stability, ability to bind to target, and the ability to selectivelypair for these heterodimers was determined as described in Example 5 andare shown in Table 11.

TABLE 11 Tm (° C.) TF Binding KD (nM) H1 Readout H2 Readout Design H1-L1Tm H2-L2 Tm H1-L1 H2-L2 H1-L1 H1-L2 H2-L2 H2-L1 26 67.8 73.6 92 1 106 127 71.0 67 1 85 12 28 85 4 94 1 29 64.1 68.3 93 1 102 1 30 67.0 75.50.024 0.060 96 1 102 1 31 74.3 0.025 106 1 78 1 32 98 1 93 1

Wild-type anti-TF D3H44 Fab KD=0.052 nM; Wild-type anti-TF Fab Tm=˜76°C.

Example 9: Additional Heterodimers

The following heterodimer pairs were also prepared and tested for theirability to selectively pair.

TABLE 12 Design Designs Region Strategy Heavy Chain 1 Light Chain 1Heavy Chain 2 Light Chain 2 ZW #1 Variable Steric Q39R Q38E V37W F98A ZW#2 Variable Combo — F98W V37W F98A ZW #3 Variable Combo Q39R Q38EV37W_Q39E Q38R_F98A ZW #4 Variable Combo V37I_Q39R Q38D_F98W V37W_Q39EQ38R_F98A ZW #5 Variable Combo V37I_Q39D Q38R_F98W V37W_Q39R_W107FQ38E_F98L ZW #6 Constant Electrostatic D148G_Q175R Q124E_Q160E_T178DK147T_Q175D_S183L Q160K_T178R ZW #7 Constant Electrostatic L145K_D148GQ124E_V133D L145E_K147T Q124R ZW #8 Constant Electrostatic D148G_Q175KQ124E_Q150E_T180E L145E_K147T Q124R_Q160K_T178R ZW #9 ConstantElectrostatic K147L_Q175E S131K D148G_Q175K Q124E_Q160E_T180E ZW #10Constant Electrostatic S181R Q124E_Q150E_T178D K147L_Q175E S131K

Residue numbering in Table 12 follows the convention used for residuesin the crystal structure of D3H44 Fab (PDB ID=1JPT [Faelber K et al., J.Mol. Biol. (2001) 313:83-97];www.rcsb.org/pdb/explore/explore.do?structureId=1JPT).

TABLE 13 Design H1_mutation L1_mutation H2_mutation L2_mutation 4691S181R Q124E_Q160E_T180E K147L_Q175E S131K 4686 D148G_Q175KQ124E_Q160E_T180E L145E_K147T Q124R_Q160K_T178R 5838 Q39E_S181RQ38R_Q124E_Q160E_T180E Q39R_K147L_Q175E Q38E_S131K 5826 Q39E_S181RQ38R_Q160E_T180E Q39R_K147T_Q175E Q38E_S131K Design 1 L145K_D148GQ124E_V133D L145E_K147T Q124R Design 2 D148G_Q175K Q124E_Q160E_T180EL145E_K147T Q124R_Q160K_T178R Design 3 S181R Q124E_Q160E_T178DK147L_Q175E S131K K147L_Q175E S131K D148G_Q175K Q124E_Q160E_T180E Design4 V37I_Q39R Q38D_F98W V37W_Q39E Q38R_F98A Design 5 Q39R Q38E V37W_Q39EQ38R_F98A Design 6 V37I_Q39D Q38R_F98W V37W_Q39R_W107F Q38E_F98L

Residue numbering in Table 13 follows the convention used for residuesin the crystal structure of D3H44 Fab (PDB ID=1JPT [Faelber K et al., J.Mol. Biol. (2001) 313:83-97];www.resb.org.pdb/explore/explore.do?structureId=1JPT).

Example 10: Assessment of Preferential Pairing of Heterodimers inCo-Expression Sets Comprising Constant Domain and/or Variable DomainModifications in D3H44 Heavy and Light Chain Fab Format

Co-expression sets in addition to those shown in FIGS. 1 and 2 weredesigned. Constructs encoding the D3H44 IgG heavy and light chains inFab format comprising amino acid modifications according to the designof the co-expression set were prepared as described in Example 1. Theability of the D3H44 heavy and light chain Fab pairs to preferentiallypair was assessed as described in Examples 2 and 3. The stability andbinding affinity of the designs was determined as described in Example5. The results shown in Tables 14 and 15 are cumulative and includeresults for designs shown in FIGS. 1 and 2 in addition to new designs.The amino acid modifications shown in these tables are identified withreference to the amino acid sequence of D3H44 heavy chain and D3H44light chain. See Tables A, A1, and A2.

Note that “Design” or “Design set” in this application is referring to aset of mutations on H1, L1, H2, and L2 chains. “LCCA design” refers toset of mutations in H1, L1 and L2.

Each unique set of H1, L1 and L2 mutations (LCCA format) was assigned aunique number, or so called ‘unique identifier’. When data is presentedin H1 L1 H2 L2 format (Fab pair format or SMCA), such a design set isconsequently denoted with a ‘unique identifier set’ comprised of uniqueidentifiers for the two constituent LCCAs (e.g. 1-2). Designs featuredin the D3H44 LCCA data set were assigned first. Designs that are notpresent in this set, but are present in different homogeneous or mixedsystem data or/and in different formats (MCA) are in addition denotedwith *. In this exercise of assigning unique identifiers, via automaticprocessing of numerous tables, some redundancy has arisen. Cases wheredifferent WT amino acids in systems other than D3H44 occupy the sameposition are: 309*=319*=47, 316*=101, 317*=183, 318*=182, 310*=48,311*=102, 323*=180, 324*=179. Cases where additional mutations wereincorporated for LC/MS are: 442*=326*, and 443*=23.

Note that the majority of LCCA experiments were performed on constructslacking interchain Fab disulfide bond(s) located in the constant domain(H/C233S-L/C214S).

In Table 14 provided are designs that exhibit correct pairingspecificity of 55%: 45% (H1-L1:H1-L2 and H2-L2:H2-L1) or greater. Forthe purposes of highlighting a particular design's success with respectto preferential pairing, two complementary LCCA sets (H1, L1, L2 and H2,L2, L1) are represented in a pair Fab format.

Presence of tags (L: HA and FLAG and H: ABD2) does not affect theexpected neutral pairing of ˜50%: 50% for D3H44 WT (this is furthersupported by pairing results for the actual designs; hence taginformation is not included in this table).

In the table, the measured amount of relevant Fab species (H1-L1, H1-L2and that of H2-L2, H2-L1) was included in ratio format (H1-L1:H1-L2 andH2-L2:H2-L1). In the majority of cases, several LCCA experimentalrepeats were performed (screening and verification). A summary column inthe format of a normalized ratio (i.e. to 100% H1-L1 and H1-L2 sum) forthe median H1-L1:H1-L2 and H2-L2:H2-L1 is also provided.

Data was clustered according to thermal stability data (Tm) and antigenaffinity (in table buckets, the ‘TF’ notation is used for tissue factoraffinity) categories:

Tm1 (x=>71° C.); Tm2 (71° C.>x=>66° C.); Tm3 (66° C.>x). Tm3 categoryalso includes ND (experiment not performed) cases.

For reference, the Tm of D3H44 WT Fab (without disulfide bond) is ˜76°C.

TF1 (x=<5×KD of WT median value); TF2 (5<x=<20×KD of WT median value);and miscellaneous category that includes cases of x>20×KD of WT medianvalue where NB cases (no binding: for KD greater than 500 nM, which is˜10000×KD of WT median) were labeled separately. This last category alsoincludes ND cases (experiment not performed).

For reference, the median KD of D3H44 WT Fab is 0.06 nM (with a range of0.1).

Note: In Table columns referring to antigen affinity and thermalstability, range (min-max reading) is indicated if the number ofexperiments performed was greater than 1 (n>1).

Within each bucket, designs are ordered in descending pairingspecificity of H1-L1:H1-L2 followed by that of H2-L2:H2-L1.

An example of reading bucket categories in the table:

Tm1 only (both H1-L1 and H2-L2 Tm belong to Tm1 category)

Tm1/Tm2 (H1-L1 or H2-L2 Tm belongs to Tm1 and the other to Tm2 category)

The same logic applies to ‘TF’ categories.

An additional set of LCCA results (Table 15), also presented in the Fabpair format, obtained following an additional design cycle is includedin a separate table. This set of data is arranged in the order ofdecreasing pairing specificity and contains somewhat limited data withrespect to thermal stability.

Results in Table 14 and 15 demonstrate that our in silico designapproach led to achievement of preferential pairing of H1-L1 over H1-L2and that of H2-L2 over H2-L1 across a diverse set of designs and theirvariations. These designs generally fell into two main categories:electrostatic (based on specificity drivers that utilize hydrogenbonding or charge-charge interactions) and steric complementarity.Specificity of such pairing ranged from moderate to 100% correct pairingfor both LCCA designs. As evident from the table, some of these designsdid not impact thermal stability (Tm) or antigen binding affinity, whilesome exhibited various degrees of impact on these two properties.Furthermore, successful designs were present in both constant andvariable domain, as well as in domain design combination formats.

Example 11: Assessment of Preferential Pairing of Heterodimers inCo-Expression Sets Comprising Constant Domain and/or Variable DomainModifications in Mixed Ab or Pure Ab Heavy and Light Chain Fab Format

Certain designs described in the previous examples were tested in asystem where the heterodimer pairs were derived from a different Ab (cf.to D3H44) or two different antibodies, to assess whether the design ofthe co-expression set resulted in preferential pairing in these types ofsystems. A number of different systems were tested. In one example, oneheterodimer pair was derived from D3H44 heavy and light chains in theFab format and the second heterodimer pair was derived from pertuzumabheavy and light chains in the Fab format. In another example, oneheterodimer pair was derived from D3H44 heavy and light chains in theFab format and the second heterodimer pair was derived from trastuzumabheavy and light chains in the Fab format.

Constructs encoding the D3H44 IgG, pertuzumab, and trastuzumab heavy andlight chains in Fab format comprising amino acid modifications accordingto the design of the co-expression set were prepared as described inExample 1. The base DNA sequence for the heavy chain of pertuzumab, thebase DNA sequence for the light chain of pertuzumab, the base DNAsequence for the heavy chain of trastuzumab, and the base DNA sequencefor the light chain of trastuzumab are shown in Tables A, A1, and A2.Amino acid modifications were introduced into these sequences by sitedirected mutagenesis, or the DNA sequences were synthesized includingthe amino acid modifications from the base sequences as described inExample 1.

The ability of the heterodimer designs to preferentially pair wasassessed as described in Examples 2 and 3, except for the fact that whenmixed systems were tested the non-obligate chain employed belonged to adifferent Fab.

The results are shown in Table 16. The amino acid modifications shown inTable 16 are identified with reference to the amino acid sequence ofD3H44 heavy chain and D3H44 light chain; pertuzumab heavy chain andpertuzumab light chain; trastuzumab heavy chain and trastuzumab lightchain. See Tables A, A1, and A2.

A representative and diverse subset of designs, that exhibitedsuccessful preferential pairing in D3H44 system, were tested indifferent systems (Trastuzumab (TRAS) and Pertuzumab (PERT), as well asin mixed systems (D3H44/TRAS, D3H44/PERT and TRAS/PERT) (as with D3H44LCCA, constructs lacked Fab disulfide).

Data is presented in both LCCA (Table 16) and Fab pair formats (Table17). LCCA data reflects the minimal ‘competition unit’ (i.e.H1-L1:H1-L2) and is the optimal format for interpreting whether an LCCAdesign can successfully transfer across Fabs. Analysis in the Fab pairformat including the second Fab pair (i.e. H2-L2:H2-L1) furtherillustrated the degree of translation into whole design (i.e. H1-L1 andH2-L2) and its efficacy in these different Fab systems. Apart from theratio of H1-L1:H1-L2, we present the relative propensity for correctpairing relative to incorrect pairing as a scalar, whereScalar=ln(H1-L1:H1-L2).

In some of the mixed systems, an inherent cross-system pairingpreference (e.g. H_D3H44 pairs preferentially with L_PERT than L_D3H44)was observed (Table 17). In the same example, H_D3H44 preferentialpairing with L_PERT was also supported by thermal stability measurements(Tm), which indicated that H_D3H44-L_PERT species was more stable thanH_D3H44-L_D3H44. Hence, along with reporting the actual species amountsmeasured, the normalized data to respective WT LCCA system (REF)behavior is presented in the form of ΔScalar(VAR-REF_WT) whereΔScalar=ln (H1-L1:H1-L2/H2-L2:H2-L1). This metric is an indicator of theactual effectiveness of the LCCA design. Data included in the tablescomprise designs that yielded an equivalent of 55%: 45% or greaterparing specificity (i.e. ΔScalar (VAR-REF_WT)>0.2).

Unlike in the D3H44 system, certain species ratios appear to be at timesaffected by the light chain tag in WT PERT only and WT TRAS (to a lesserdegree) systems (Table 18). This appears to be due to random events ofHA-tag cleavage (LC/MS evidence available when systems are tested in Mabformat), rather than tag interference with pairing. Hence tags weretaken into account when presenting results in the relevant tables.

A summary of the results, in both LCCA and Fab pair format, across thedifferent systems is reported in Tables 19 and 20. The results indicatedesign transferability across different tested Fab systems. Theseresults do not necessarily reflect that some of the designs are betterthan others; nor do they reflect a more comprehensive transferabilityestimate. Successful LCCA designs (e.g., median ΔScalar(VAR-REF_WT)>0.2) in two systems or more, presented in Table 19,constitute app. 30% of tested LCCA designs in these different systems.This is indicative of transferability, dependent on the particularsystem and design. Thus, having a collection of unique designs allowedfor the creation of bispecific pairs for a number of systems andhighlights the utility of a library of design set that can be evaluatedin the context of any antibody (or antibody pair) of interest. Thisexample indicates that mutation/design sets can be used to achievepreferential pairing of heterodimers in co-expression sets comprisingconstant domain and/or variable domain modifications in mixed Ab or pureAb heavy and light chain Fab format.

Example 12: Assessment of Preferential Pairing of Heterodimers inCo-Expression Sets Comprising Constant Domain and/or Variable DomainModifications in D3H44 Heavy and Light Chain, in Full-Length Heavy Chain(Mab) Format

The heterodimer co-expression set designs were assessed to determine ifthey also allowed for preferential pairing in a format (Mab format)where the heavy chain is a full-length heavy chain and not a Fabportion.

Preparation of Constructs:

Constructs encoding the D3H44 IgG heavy and light chains comprisingamino acid modifications according to the design of the co-expressionset were prepared as follows. D3H44 Fab light chain constructs wereprepared as described in Example 1. D3H44 heavy chain sequences wereprepared as described in Example 1, except a full-length D3H44 heavychain was created by appending the IgG1*01 DNA sequence, encoding thehinge-CH2-CH3 domains, onto the C-terminus of the D3H44 Fab heavy chain.Of note, the canonical C-terminal heavy chain lysine residue was removedin order to prevent LC-MS signal heterogeneity due to C-terminal lysineclipping (Lawrence W. Dick Jr. et al., Biotechnol. Bioeng. (2008)100:1132-43).

Mab Assay Format

The ability of the D3H44 heavy and light chains to preferentially pairwas assessed as follows: One full-length D3H44 heavy chain construct wasco-expressed with two unique D3H44 light chain constructs, yieldingthree possible antibody species: H1-L1:H1-L1, H1-L2:H1-L2 andH1-L1:H1-L2 (see FIG. 10). The relative light chain pairing specificityin terms of the amount of preferred H1-L1:H1-L1 species vs. others wasdetermined using LC-MS after proteinA (pA) purification. Where possible,chains were left untagged, provided the three possible Mab speciesresulting from co-transfection of the three chains differed by at least50 Da from each other. When mass differences precluded this possibility,at least one of the light chains was constructed with an N-terminal HAor FLAG tag fusion in order to provide sufficient mass differentiationbetween species. As described in Example 2, heavy chain was kept inlimiting quantities (i.e. HC<L1+L2).

Transfection Method for Mab Assay Format

Co-expression sets comprising one heavy chain and two light chainconstructs prepared as described in Example 12 were transfected intoCHO-3E7 cells as follows. CHO-3E7 cells, at a density of 1.7-2×10⁶cells/ml, were cultured at 37° C. in FREESTYLE™ F17 medium (Invitrogencat #A-1383501) supplemented with 4 mM glutamine and 0.1% Pluronic F-68(Invitrogen cat #24040-032). A total volume 50 ml were transfected witha total of 50 ug DNA using PEIPRO® (Polyplus cat #115-010) at a DNA:PEIratio of 1:2.5. Twenty-four hours after the addition of the DNA-PEImixture, the cells were transferred to 32° C. Supernatants were testedfor expression on day 7 by non-reducing SDS-PAGE analysis followed byCoommassie blue staining to visualize the bands. HC:L1:L2 ratios usedwere 1:1:1.

Mass Spectrometry Method for Mab Assay Format

The degree of preferential D3H44 light chain pairing to D3H44 heavychain in co-expression sets was assessed using mass spectrometry afterprotein A purification and deglycosylation The purified samples werede-glycosylated with PNGaseF as follows: 0.2 U PNGaseF/μg of antibody in50 mM Tris-HCl pH 8.0, overnight incubation at 37° C., final proteinconcentration was 0.45 mg/mL. The protein samples were analyzed by LC-MSusing an Agilent 1100 HPLC system coupled to an LTQ-Orbitrap XL hybridmass spectrometer (ThermoFisher Scientific) via a high-flow electrosprayinterface. The samples (2.5 g) were injected onto a 2.1×10 mm POROS™ R2column (Applied Biosystems) and eluted using the following gradientconditions: 0-3 min: 20% solvent B; 3-6 min: 20-90% solvent B. Solvent Awas 0.1% formic acid aq. and solvent B was 65% ACN, 25% THF, 9.9% ddH₂O,0.1% formic acid. The flow rate was 1 mL/min. The flow was splitpost-column to direct 100 L/min into the electrospray interface. Thecolumn and solvent were heated to 80° C. to improve protein peak shape.The LTQ-Orbitrap XL was calibrated using ThermoFisher Scientific's LTQPositive Ion ESI calibration solution (caffeine, MRFA and ULTRAMARK®1621), and tuned using a 10 mg/mL solutions of CsI. The cone voltage(source fragmentation setting) was 40 V, the FT resolution was 7,500 andthe scan range was m/z 400-4,000.

The protein spectra were deconvoluted using the MaxEnt module of theMassLynx instrument control and data analysis algorithm (Waters).Briefly, the raw protein LC-MS data were first opened in QualBrower, thespectrum viewing module of Xcalibur (Thermo Scientific) and converted tobe compatible with MassLynx using Databridge, a file conversion programprovided by Waters. The converted protein spectra were viewed in theSpectrum module of MassLynx and deconvoluted using MaxEnt. Theabundances of the different antibody species in each sample weredetermined directly from the resulting molecular weight profiles.

The results are shown in Table 21. The amino acid modifications shown inTable 21 are identified with reference to the amino acid sequence ofD3H44 heavy chain and D3H44 light chain. See Tables A, A1, and A2.

A subset of designs that exhibited successful preferential pairing inD3H44 system, which are also representatives of a diverse design set,were chosen for assessment of format transferability, i.e., from LCCAwith Fab structures to Mab competition assay (MCA) based on Mabstructures.

As shown in Table 21, D3H44 WT used in a Mab competition assay exhibiteddeviation from the expected theoretical species distribution of 50%H1-L1_H1-L2 and 25% of each H1-L1_H1-L1 and H1-L2_H1-L2, where lightchains were distinguished by the presence or absence of a tag (HA orFLAG). This deviation is likely a result of tag dependent expressionlevels rather than tag dependent pairing (e.g., based on experimentsperformed at different WT H1:L1:L2 ratios, as well as indirectobservation on the basis of design behavior). Median values for all themeasured species across experimental repeats and/or variants differingby tag identity were included as well.

An example of LC/MS spectra of two successful Mab assay variants (FIG.11) that comprise a given design are found in FIGS. 12A-12B. In the caseof these variants a majority of the measured species corresponded to thedesired H1-L1_H1-L1 species. Expression profiles, UPLC-SEC (shown onlyfor H2-L2_H2-L2 species (in the Fig. denoted as H2-L2)), and DSCthermograms reported in FIGS. 13A-13C are typical for characterizationof the variants carried out for certain hits. In this particular casethese demonstrated well-expressed, homogeneous species with relativelyminor impact on Fab stability.

The results in Table 21 demonstrate that transferability into the Mabformat was achieved with notable success among the LCCA designs. 11 outof the 12 LCCA designs tested exhibited equal to or greater than thetheoretical 25% correctly paired species. This data indicates that theLCCA format used as an initial screen of design library is suitable forassessing and/or predicting design success in the Mab format.

Example 13: Assessment of Preferential Pairing of Heterodimers inCo-Expression Sets Comprising Constant Domain and/or Variable DomainModifications in a Bi-Specific Antibody Format

The heterodimer co-expression set designs were assessed to determine ifthey also allowed for preferential pairing in a bi-specific Mab antibodyformat. In this example, the Fc region of the full-length heavy chain ofeach heterodimer was asymmetrically modified to promoteheterodimerization of the unique heavy chains compared tohomodimerization.

Preparation of Constructs:

The heterodimer co-expression set designs were tested in the context ofthe following bi-specific antibodies: a) D3H44/pertuzumab, b)D3H44/trastuzumab, c) D3H44/ramucirumab, and d) trastuzumab/ramucirumab.The D3H44, pertuzumab, and trastuzumab heavy and light chains comprisingamino acid modifications in the constant and/or variable domains wereprepared as described in Example 12, except complementary Fcheterodimerization mutations were introduced into each two heavy chainof a co-expression design set. The ramucirumab heavy and light chainswere prepared based on the base DNA sequence for the ramucirumab heavychain and light chain. See Table A. The CH3 sequences of the heavychains included the following amino acid modifications:

a) D3H44/pertuzumab: Chain A: T371V_T389L_K420L_T422W, Chain B:T371V_L372Y_F436A_Y438V

b) D3H44/trastuzumab: Chain A: T371V_T389L_K420L_T422W, Chain B:T371V_L372Y_F436A_Y438V

c) D3H44/ramucirumab: Chain A: T371V_T389L_K420L_T422W, Chain B:T371V_L372Y_F436A_Y438V

d) Trastuzumab/ramucirumab: Chain A: T371V_T389L_K420L_T422W, Chain B:T371V_L372Y_F436A_Y438V

Assay Format (SMCA)

The ability of the heterodimer co-expression set designs topreferentially pair to form a bi-specific antibody was assessed asdescribed below. The assay is based on co-expressing the four chains(two from Ab1 and two from Ab2) and detecting the presence of correctlyformed bispecific antibody using mass spectrometry (LC-MS). The assaywas carried out as follows. FIG. 14 provides a schematic depicting thefour starting polypeptide chains and the potential products resultingfrom co-expression of these starting polypeptide chains in the absenceof preferential pairing between heavy and light chains of theheterodimer pairs. Two full-length heavy chain constructs wereco-expressed with two unique light chain constructs, yielding tenpossible antibody species: H1-L1:H1-L1, H1-L2:H1-L2, H1-L1:H1-L2,H2-L1:H2-L1, H2-L2:H2-L2, H2-L1:H2-L2, H1-L1:H2-L1, H1-L2:H2-L2,H1-L2:H2-L1 and H1-L1:H2-L2. The latter species is the correctly pairedheterodimer (see Fig. below). The relative pairing specificity in termsof amount of preferred species H1-L1:H2-L2 vs. others was determinedusing LC-MS after pA purification. When possible, chains were leftuntagged, provided all Mab and half-Ab species differed from each otherby at least 50 Da. When mass differences precluded this possibility, oneof the light chains was constructed with an N-terminal HA tag fusion inorder to provide sufficient mass differentiation between species. Werefer to this assay involving the expression and screening steps of abispecific antibody as SMCA.

Transfection Method

Co-expression sets comprising two heavy chains and two light chainconstructs prepared as described in Example 1 were transfected intoCHO-3E7 cells as follows. CHO-3E7 cells, at a density of 1.7-2×10⁶cells/ml, were cultured at 37° C. in FREESTYLE™ F17 medium (Invitrogencat #A-1383501) supplemented with 4 mM glutamine and 0.1% Pluronic F-68(Invitrogen cat #24040-032). A total volume of 20 ml were transfectedwith a total of 20 ug DNA using PEIPRO® (Polyplus cat #115-010) at aDNA:PEI ratio of 1:2.5. Twenty-four hours after the addition of theDNA-PEI mixture, the cells were transferred to 32° C. Supernatants weretested for expression on day 7 by non-reducing SDS-PAGE analysisfollowed by Coommassie blue staining to visualize the bands. H1:H2:L1:L2ratios used were initially kept neutral (15:15:35:35) to assessexpression efficiency. A set of H1:H2:L1:L2 DNA ratios was then testedin CHO expressions to assess which condition(s) produced a mixturereflecting the theoretical distribution of the different species whenall chains are wild-type. These ratios compensate for naturaldifferences in expression levels and/or intrinsic pairing biases betweenheavy and light chains of the two different antibodies.

SPR Biosensor Assays

EDC: I-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride; sNHS:N-hydroxysulfosuccinimide; SPR: surface plasmon resonance; EDTA:ethylenediaminetetraacetic acid; HEPES:4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; TF: tissue factor.

SPR supplies. GLC sensorchips, the Biorad PROTEON™ amine coupling kit(EDC, sNHS and ethanolamine), and 10 mM sodium acetate buffers werepurchased from Bio-Rad Laboratories (Canada) Ltd. (Mississauga, ON).Recombinant Her-2 protein was purchased from eBioscience (San Diego,Calif.). PBS running buffer with 0.05% Tween20 (PBST) was purchased fromTeknoca Inc. (Hollister, Calif.). Goat polyclonal anti-human Fc antibodywas purchased from Jackson Immuno Research Laboratories Inc. (WestGrove, Pa.).

All surface plasmon resonance assays were carried out using a BioRadPROTEON™ XPR36 instrument (Bio-Rad Laboratories (Canada) Ltd.(Mississauga, ON)) with PBST running buffer at a temperature of 25° C.The anti-human Fc capture surface was generated using a GLC sensorchipactivated by a 1:5 dilution of the standard BioRad sNHS/EDC solutionsinjected for 140 s at 100 μL/min in the analyte (horizontal) direction.Immediately after the activation, a 10 ug/mL solution of anti-human Fcantibody in 10 mM NaOAc pH 4.5 was injected in the ligand (vertical)direction at a flow rate of 25 μL/min until approximately 3000 resonanceunits (RUs) were immobilized. Remaining active groups were quenched by a140 s injection of 1M ethanolamine at 100 μL/min in the analytedirection, and this also ensures mock-activated interspots are createdfor blank referencing. The screening of the antibody variants forbinding to Her2 or TF antigen targets occurred in two steps: an indirectcapture of the antibody variants onto the anti-human Fc antibody surfacein the ligand direction followed by the simultaneous injection of 5concentrations of purified antigen and one buffer blank for doublereferencing in the analyte direction. Firstly, one buffer injection for30 s at 100 uL/min in the ligand direction was used to stabilize thebaseline. For each antibody variant capture, unpurified variants incell-culture media were diluted to 4% in PBST. One to five variants orcontrols were simultaneously injected in individual ligand channels for240 s at flow 25 μL/min. This resulted in a capture of approximately 400to 600 RUs onto the anti-human Fc surface. The first ligand channel wasleft empty to use as a blank control if required. This capture step wasimmediately followed by two buffer injections in the analyte directionto stabilize the baseline, and then 60 nM, 20 nM, 6.7 nM, 2.2 nM and0.74 nM antigen (TF or Her2) along with a buffer blank wassimultaneously injected at 50 μL/min for 120 s with a 300 s dissociationphase. The captured antibody surfaces were regenerated by an 18 s pulseof 0.85% phosphoric acid for 18 s at 100 L/min to prepare for the nextinjection cycle. Sensorgrams were aligned and double-referenced usingthe buffer blank injection and interspots, and the resulting sensorgramswere analyzed using PROTEON™ Manager software v3.1. Thedouble-referenced sensorgrams were fit to the 1:1 binding model. Rmaxvalues for each antigen were normalized to antibody capture levels foreach variant and compared to 100% controls.

The stability of the bi-specific antibodies was tested as described inExample 5. The LCMS analysis of the bi-specific antibodies was carriedout using the procedure described in Example 12.

A number of selected D3H44 LCCA designs were tested in the SMCA format.Furthermore, some designs were directly evaluated in the SMCA formatonly. A majority of designs were tested in the D3H44/Pertuzumab system.A subset of designs that were selected in this system (involving avariety of designs) were further tested in three additional bispecificMab systems: D3H44/Trastuzumab, D3H44/Ramucirumab (RAMU), andTrastuzumab/Ramucirumab systems.

Inherent cross-system light/heavy chain preference observed forD3H44/Pertuzumab and D3H44/Trastuzumab in LCCA experiments (Example 11)was reproducible in the full antibody format as well. Furthermore,similar behavior, although with different cross-system pairingtendencies, was also observed for the other two systems (Table 22).

Desired bispecific species, H1-L1_H2-L2, cannot generally bedistinguished experimentally on the basis of LC/MS from the mispairedtype: H1-L2_H2-L1. As such, when bispecific content is reported in thetables, it cannot be completely excluded that it does not contain thistype of mispaired species. However, the very low content observed forspecies such as H1-L2_H1-L2 and H2-L1_H2-L1 as well as H1-L2 and H2-L1half antibodies is indicative that only minor if any contamination ofthe bispecific species occurred. All other species present in aparticular sample measured by LC/MS are included in the table. In mostcases MS peaks were accompanied by side peaks; however these wereannotated only for the bispecific species. When normalizing MS peakintensities, these adduct species were not taken into account. Thenumber represented for side peak(s) in the tables was obtained byintensity comparison with that for the main bispecific peak. Somepreliminary analysis indicated the side peak as being correlated withthe presence of light chain tags involving the formation of adducts orheterogeneity in the cleavage of leader peptides and it is likelyrepresentative of the main peak species.

Finally all paired species were summed up, in addition to mispairedspecies, to obtain the ratios reported in the paired:mispaired column.This was further used to calculate ΔScalar (VAR-REF_WT) following themathematical approach described in Example 11 to demonstrate thestrength of a particular design. Within a system, designs were orderedin descending values of the Scalar metric.

Due to the cross-system natural preference for pairing of light andheavy chains described in Example 12, DNA ratios of H1:L1:H2:L2 requiredalteration (e.g. over-expression of H1 (D3H44) over H2 (PERT)) to yieldthe highest content of desired bispecific species (H1-L1_H2-L2). Optimalratios may also vary to some degree with a particular design as aresult. DNA ratios mainly affect the actual ratio of bispecific species:paired half antibody species (usually of one type: H1-L1 or H2-L2). Anexample is included in Table 23, where DNA titration with this ratiovaried but the overall ratio of paired:mispaired species was relativelyconstant.

In the case of the D3H44/Pertuzumab system, a limited set of DNAtitrations was performed for the majority of designs. Data for ratiosthat resulted in the highest paired:mispaired species content are shownin Tables 22 and 24. For the other three systems, transfections wereperformed only at one ratio, as reported in the tables. If at thereported ratio the experiment was repeated, a mean value was included inthe table. Information with respect to tag identity was not included astag influence was not observed for the WT (Table 26). The WT referenceprovided was chosen as such to be representative of the most common DNAratio among reported design data.

LC/MS analysis was performed on samples that were stored at pH4 as wellas at pH7. Experiments on samples stored at pH7 were tested in the caseof D3H44/Trastuzumab, D3H44/Ramucirumab (RAMU), andTrastuzumab/Ramucirumab systems. Hence data is presented in two tables(Table 22 (pH4) and 24 (pH7)). Satisfactory correlation was observed forpaired:mispaired species ratios between the two experiments, indicatingthat pH did not likely play a substantial role in the LC/MS experiment,i.e., in characterization of the type of species present.

FIGS. 15A-15C presents the LC/MS analysis results of the bispecificconstruct targeting TF and Her2 based on the designs presented in FIG.11 and FIGS. 12A-12B in the MCA format. Close to 92% of the preferredbispecific antibody with the correct pairing of obligate heavy and lightchains was observed. FIG. 16 presents the expression profile of theprotein product in the supernatant and following protein A purificationin SDS PAGE as well as the SEC profile of the protein A purifiedcompound. FIGS. 17A-17B presents the bispecific target binding featuresof the bispecific molecule, first to the two targets (TF and Her2independently) and then in a sandwich (bridging) mode.

In some cases, the mutations at H/S 115 and H/S156, featured in limitedset of variants, were not part of the actual design, but rather wereadded for practical reasons to gain necessary mass difference for thepurposes of a LC/MS experiment. These amino acids are located on thesurface of the constant domain, sufficiently away from the actual designset of mutations and is not expected to impact the behavior of theantibody.

Thermal stability and antigen affinity were assessed for a number ofdesigns in the D3H4/Pertuzumab system and are presented in Table 25. Tmvalues (indicated in italics) were annotated manually. Homodimeric Mabcontrols (WT) exhibited the following stability range of thermal melting(Tm) for product expressed in transfection repeats (at pH 7): PERT(72.03-77.72) and for D3H44 (77.97-78.88). The wider range observed forpertuzumab is likely due to its intrinsic properties. Affinitymeasurements were undertaken post pA purification only. Observed KDrange for Homodimeric Mabs is: TF: 0.04-0.076 nM, HER2: 1.84-6.3 nM. Inmost cases one would expect two melting transitions due to the aboveindicated different ranges for the two Fabs. In cases where only onevalue for Tm is reported, it potentially arose due to one of tworeasons: variation of pertuzumab stability or/and destabilization of thedesign on the D3H44 Fab that may coincide with that of pertuzumab Fab.Results indicate that selection of SMCA variants ranges from the onesthat affect thermal stability minimally as well as antigen binding tothe ones that do so to varying degrees.

Some designs were tested with both possible Fc mutation placements.These are identifiable by having the same unique identifier and denotedwith *D3H44 (or in Table 25 with #preceding unique identifier set). Asevident from Table 22, placement of Fc mutations does not appear toinfluence the paired:mispaired species outcome with a limited exception.

The SMCA results show that a substantial number of designs representinga diverse set can overcome the natural cross-species pairing tendency inthe Mab format. Among these, close to a quarter of the designs testedfall into the category of high paired:mispaired ratio (>=80:20) (numbersare provided for the more exhaustively tested system, D3H44/Pertuzmab).Comparison of design effectiveness across different systems revealsvarying degrees of transferability. Furthermore, approximately 60% ofdesigns, listed in Table 27, transported from the D3H44 LCCA into theSMCA format resulted in a high degree of preferential pairing (>75%:25%) in at least one of the four tested SMCA systems.

For designs tested in systems other than D3H44/Pertuzumab, designplacement was inverted with respect to the binding domains as well, e.g.H1-L1 designs on D3H44 binding domain, H2-L2 designs on TRAS as well asH1-L1 designs on TRAS and H2-L2 designs on D3H44. The resultsdemonstrate that a design's effectiveness in a majority of cases couldbe impacted by such a flipped placement.

The results discussed above indicate that a design's transferability canbe impacted by a combination of antigen binding domains coupled with thedriving potential of design constituents (e.g. H1L1L2 driving may bebetter than H2L2L1). The results indicate that a large number of basecore designs work across a range of Mab pairs to form a bispecific Abwith greater than 75% selective pairing.

Example 14: Molecular Modeling and Computer Guided Engineering of FabInterface

We employed a structure and computational molecular modeling guidedapproach to produce a library of heavy and light chain mutation designsthat can be screened in the context of other antibodies or fragmentsthereof to identify mutations that exhibit the desired specificity inthe antibodies of interest. The design strategy for engineeringpreferential HC-LC pairing included first identifying a representativeAb (i.e. D3H44) to work with. Key criteria of this Ab are shown in Table28.

TABLE 28 Criteria Importance Human or humanized IgG1/κ Similarity Hascommonly used V_(H) and V_(L) subgroups Framework close to germlineV_(H):V_(L) interdomain packing angle close to observed average for FabsStructure available for apo- and complexed Fab Design No majorstructural changes observed upon binding antigen Antigen binding can bereadily assayed Assay

As indicated in Table 28, key criteria presented by this antibody wasthat it was a human/humanized Ab, with commonly used VH and VL subgroupsand minimal framework region mutations. In addition, structuralconsiderations were that the VH:VL interdomain angle should be close tothe average observed for Abs. After selection of the Fab, an in silicoanalysis of the Fab interface was carried with the aim being to identifyand understand the important residues. A two-pronged approach was taken.First, a global analysis of the sequence conservation across the Fabvariable and constant interfaces was carried out via sequence andstructure alignments of publicly available Abs. An alignment of constantand variable domain sequences from various antibody subgroups is shownin FIGS. 6A-6E. In parallel, the crystal structure interface D3H44 wasanalyzed using a number of molecular modeling tools listed in FIG. 18(e.g. RESIDUECONTACTS™). These analyses resulted in the identificationof a list of hotspot positions for engineering preferential HC-LCpairing. The hotspot positions determined from this analysis are listedin Table 29.

TABLE 29 Hotspot amino acid positions at the interface of the heavy (H)and light (L) chain in a typical Fab derived from human VH and VL kappachains. These positions and amino acids are also mostly conserved in theVL lambda chains. These are mainly framework residues except for a fewlocated in the CDR3 loops. The amino acids in the parent D3H44 sequenceswith Kabat numbering are provided in Tables A1-A2. H (Kabat) L (Kabat)V37 Y36 Q39 Q38 L45 P44 W47 L89 F100 F98 W103 F116 L124 F118 A139 V133F174 L135

Next, potential mutations and designs at the hotspot positions as wellas positions neighboring the hotspots of interest in the 3D crystalstructure were simulated and identified via in silico mutagenesis andpacking/modeling with ZYMEPACK™ and scored on the basis of a number offactors including steric and electrostatic complementarity. FIG. 11presents a limited number of hotspot positions at the heavy and lightchain interface in the variable domains and how mutations can beintroduced at these interface positions to facilitate selective pairingof the obligate chains while disfavoring the formation of incorrectchain pairs. Steric complementarity was modeled and also computed on thebasis of energy factors such as van der Waals packing, cavitationeffects and close contact of hydrophobic groups. Similarly,electrostatic interaction energies were modeled and evaluated on thebasis of coulomb interactions between charges, hydrogen bonds, anddesolvation effects. Both the preferred heavy and light chain pairmodels such as H1:L1 (or H2:L2) and incorrect pair such as H1:L2 (andH2:L1) obtained by introducing the mutations of interest were simulatedto compute the relative steric and electrostatic scores. This allowed usto decide if a particular mutation set lead to favorable energies i.e.greater steric or electrostatic complementarity for the preferred(obligate) heavy-light chain pairs relative to the incorrect(non-obligate) pairs. The computed steric and electrostatic energies arecomponents of the free energy associated with the light and heavy chainpairing. Hence greater steric and electrostatic complementarity isindicative of a larger free energy change associated with the pairing ofthe obligate pair relative to the pairing of the non-obligate pair. Thegreater steric or electrostatic complementarity results in preferential(selective) pairing of the obligate heavy and light chains relative tothe non-obligate pair and can be detected in terms of the percentage ofthe two products (obligate paired vis-à-vis the non-obligate pairedheavy and light chain) upon co-expression. The greater steric orelectrostatic complementarity in the obligate pair can also be typicallyobserved in terms of better/greater thermal stability relative to thenon-obligate pair. Candidate designs were shortlisted and ranked.Designs were initially tested in vitro using the LCCA system. Moderatelyperforming designs displaying non-optimal biophysical characteristicssuch as poor HC-LC pairing specificity, low thermal stability, orreduced antigen binding affinity, were further improved via additionalrounds of in silico design and in vitro screening. The best designs werethen tested in a bispecific Mab format; in vitro screening primarilybeing via the SMCA format.

Example 15: Generation of Bispecific Antibody Given Mab1 and Mab2 Usinga Library of Bispecific Antibody Mutation Design Sets

In one embodiment, presented here is a bispecific antibody mutationdesign set aimed at selectively forming bispecific antibodies given twocanonical antibodies Mab1 and Mab2 comprising of the antigen bindingfragments Fab1 and Fab2 respectively. The design set consists of cognatemutations corresponding to Fab1, Fab2 and Fc respectively. Mutations areintroduced at the interface of light and heavy chain of Fab1 to achieveselective pairing between the two obligate chains in the presence ofcompeting light and heavy chain of Fab2. Selective pairing is achievedby introducing favorable complementary mutations in the two obligatelight and heavy chains on the basis of steric, hydrophobic orelectrostatic complementarity between certain hotspot framework residuesat the interface while involving these mutated residues in unfavorableinterface interaction for the non-obligate chain pairs. In each designset selective pairing mutations can also be introduced at the interfaceof light and heavy chain of Fab2 to achieve selective pairing betweenthese two obligate chains in the presence of competing light and heavychain of Fab1. The mutations are aimed at reducing the mis-pairing oflight chain from Fab1 with heavy chain of Fab2 and vice-versa. Mutationsare introduced at the Fc interface in order to achieve selective pairingof heavy chains to form asymmetric antibody molecules comprising twodifferent heavy chains.

Engineering at certain interface residue positions of light and heavychains of an antibody can often lead to detrimental effects such as lossin antigen binding affinity, stability, solubility, aggregationpropensity etc of that antibody. A number of related properties can beaffected such as kon and koff rates, melting temperature (Tm), stabilityto stress conditions like acid, base, oxidation, freeze/thaw, agitation,pressure etc. This is often impacted by the complementarity determiningregions (CDR's) of the antibody of interest. Given that the CDR's ofantibodies are not generally the same, the impact of the mutation designset may not be the same across all antibodies. In another embodiment, anumber of different bispecific mutation design sets constituting alibrary of bispecific antibody mutation design sets are defined thatinvolve mutations at different hotspot positions at the interface. Alibrary of such bispecific antibody mutation design sets is shown inTable 30. Presented here is a method to create a bispecific antibodywith noted purity relative to other contaminants containing incorrectlypaired antibody-like structures, given any two available antibodies Mab1and Mab2. The light and heavy chains of Mab1 and Mab2 are co-expressedafter introducing the cognate mutations of each of the mutation designsets and the expressed antibody product is analytically screened toestimate the purity of the preferred bispecific antibody relative toother Mab like species expressed in the protein product. In someembodiments the analytical screening procedure may be based on an LC-MStechnique. In some embodiments the analytical screening procedure may bebased on charge based separation such as a capillary isoelectricfocusing (cIEF) technique. An example of the screening technique ispresented in Example 13 based on the SMCA procedure. In some embodimentsthe noted purity of the bispecific antibody is defined as being greaterthan 70% of all the obtained Mab like species in the expressed proteinproduct. In some embodiments the noted purity of the bispecific antibodyis defined as being greater than 90% of all the obtained Mab likespecies in the expressed protein product. The procedure for preparationand selection of bispecific Mab design set given Mab1 and Mab2 is shownschematically in FIG. 19A-19C.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated by reference into thespecification to the same extent as if each individual publication,patent or patent application was specifically and individually indicatedto be incorporated herein by reference.

While the invention has been particularly shown and described withreference to a preferred embodiment and various alternate embodiments,it will be understood by persons skilled in the relevant art thatvarious changes in form and details can be made therein withoutdeparting from the spirit and scope of the invention.

All references, issued patents and patent applications cited within thebody of the instant specification are hereby incorporated by referencein their entirety, for all purposes.

TABLE A SEQUENCES SEQ ID NO DESCRIPTION SEQUENCE 4 D3H44 lightDIQMTQSPSSLSASVGDRVTITCRASRDIKSYLNWYQQKPGKAPKVLIYYATSLAE chain (DomainGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCLQHGESPWTFGQGTKVEIKRTVAA boundaries:PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS VL; D1-KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC K107, CL; R108-C214) 5Pertuzumab DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTlight chain GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTEGQGTKVEIKRTVAA(Domain PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSboundaries: KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC VL; D1-K107, CL; R108-C214) 6 TrastuzumabDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYS light chainGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTEGQGTKVEIKRTVAA (DomainPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS boundaries:KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC VL; D1- K107, CL;R108-C214) 7 RamucirumabDIQMTQSPSSVSASIGDRVTITCRASQGIDNWLGWYQQKPGKAPKLLIYDASNLDT light chainGVPSRFSGSGSGTYFTLTISSLQAEDFAVYFCQQAKAFPPTEGGGTKVDIKGTVAA (DomainPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS boundaries:KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC VL; D1- K107, CL;G108-C214) 8 D3H44 heavyEVQLVESGGGLVQPGGSLRLSCAASGFNIKEYYMHWVRQAPGKGLEWVGLIDPEQG chain (DomainNTIYDPKFQDRATISADNSKNTAYLQMNSLRAEDTAVYYCARDTAAYFDYWGQGTL boundaries:VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH VH; E1-TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH S113, CH1;TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD A114-K223,GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI Hinge; E226-SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK P243, CH2;TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG A244-K360,CH3; G361- G477) 9 PertuzumabEVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSG heavy chainGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQG (DomainTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG boundaries:VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK VH; E1-THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY S113, CH1;VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK A114-K223,TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN Hinge; E226-YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG P243, CH2;A244-K360, CH3; G361- G477) 10 TrastuzumabEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARTYPTNG heavy chainYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQ (DomainGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS boundaries:GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD VH; E1-KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW S113, CH1;YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE A114-K223,KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN Hinge; E226-NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP P243, CH2; GA244-K360, CH3; G361- G477) 11 RamucirumabEVQLVQSGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSS heavy chainYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVTDAFDIWGQGTMV (DomainTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT boundaries:FPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT VH; E1-CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG S113, CH1;VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS A114-K223,KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT Hinge; E226-TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG P243, CH2;A244-K360, CH3; G361- G477) 12 Trastuzumab_HCGCCACCATGGCCGTGATGGCTCCTAGAACCCTGGTGCTGCTGCTGTCTGGAGCTCTGGCTCTGACTCAGACCTGGGCTGGAGAGGTGCAGCTGGTGGAAAGCGGAGGAGGACTGGTGCAGCCAGGAGGATCTCTGCGACTGAGTTGCGCCGCTTCAGGATTCAACATCAAGGACACCTACATTCACTGGGTGCGACAGGCTCCAGGAAAAGGACTGGAGTGGGTGGCTCGAATCTATCCCACTAATGGATACACCCGGTATGCCGACTCCGTGAAGGGGAGGTTTACTATTAGCGCCGATACATCCAAAAACACTGCTTACCTGCAGATGAACAGCCTGCGAGCCGAAGATACCGCTGTGTACTATTGCAGTCGATGGGGAGGAGACGGATTCTACGCTATGGATTATTGGGGACAGGGGACCCTGGTGACAGTGAGCTCCGCCTCTACCAAGGGCCCCAGTGTGTTTCCCCTGGCTCCTTCTAGTAAATCCACCTCTGGAGGGACAGCCGCTCTGGGATGTCTGGTGAAGGACTATTTCCCCGAGCCTGTGACCGTGAGTTGGAACTCAGGCGCCCTGACAAGCGGAGTGCACACTTTTCCTGCTGTGCTGCAGTCAAGCGGGCTGTACTCCCTGTCCTCTGTGGTGACAGTGCCAAGTTCAAGCCTGGGCACACAGACTTATATCTGCAACGTGAATCATAAGCCCTCAAATACAAAAGTGGACAAGAAAGTGGAGCCCAAGAGCTGTGATAAGACCCACACCTGCCCTCCCTGTCCAGCTCCAGAACTGCTGGGAGGACCTAGCGTGTTCCTGTTTCCCCCTAAGCCAAAAGACACTCTGATGATTTCCAGGACTCCCGAGGTGACCTGCGTGGTGGTGGACGTGTCTCACGAGGACCCCGAAGTGAAGTTCAACTGGTACGTGGATGGCGTGGAAGTGCATAATGCTAAGACAAAACCAAGAGAGGAACAGTACAACTCCACTTATCGCGTCGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGGAAGGAGTATAAGTGCAAAGTCAGTAATAAGGCCCTGCCTGCTCCAATCGAAAAAACCATCTCTAAGGCCAAAGGCCAGCCAAGGGAGCCCCAGGTGTACACACTGCCACCCAGCAGAGACGAACTGACCAAGAACCAGGTGTCCCTGACATGTCTGGTGAAAGGCTTCTATCCTAGTGATATTGCTGTGGAGTGGGAATCAAATGGACAGCCAGAGAACAATTACAAGACCACACCTCCAGTGCTGGACAGCGATGGCAGCTTCTTCCTGTATTCCAAGCTGACAGTGGATAAATCTCGATGGCAGCAGGGGAACGTGTTTAGTTGTTCAGTGATGCATGAAGCCCTGCACAATCATTACACTCAGAAGAGCCTGTCCCTGTCTCCCGGCTGA 13 Trastuzumab_LCGCCACTATGGCTGTGATGGCCCCTAGGACCCTGGTGCTGCTGCTGTCCGGAGCTCTGGCTCTGACTCAGACCTGGGCTGGAGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGACGTTAACACCGCTGTAGCTTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATTCTGCATCCTTTTTGTACAGTGGGGTCCCATCAAGGTTCAGTGGCAGTCGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGCATTACACTACCCCACCCACTTTCGGCCAAGGGACCAAAGTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAAGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTGA 14 Ramucirumab_HCGCCACCATGGCCGTGATGGCTCCTAGAACACTGGTCCTGCTGCTGTCAGGGGCACTGGCACTGACTCAGACTTGGGCTGGGGAGGTCCAGCTGGTCCAGTCCGGAGGAGGACTGGTGAAGCCTGGAGGGAGTCTGCGACTGTCATGCGCCGCTAGCGGGTTCACCTTTAGCTCCTACAGCATGAACTGGGTGCGACAGGCACCAGGCAAAGGACTGGAATGGGTGTCTAGTATCTCAAGCTCCTCTAGTTACATCTACTATGCAGACAGCGTGAAGGGCCGGTTCACCATCAGCAGAGATAACGCCAAAAATTCCCTGTATCTGCAGATGAACAGCCTGCGAGCCGAGGACACCGCTGTCTACTATTGCGCACGGGTGACAGACGCCTTCGATATTTGGGGACAGGGCACCATGGTCACAGTGTCAAGCGCCTCCACCAAGGGACCAAGCGTGTTCCCACTGGCTCCATCCTCTAAAAGCACTTCCGGAGGAACCGCAGCCCTGGGATGTCTGGTGAAGGATTACTTCCCAGAGCCCGTCACAGTGTCATGGAACAGCGGGGCTCTGACCTCTGGAGTCCACACATTTCCAGCAGTGCTGCAGAGTTCAGGACTGTACAGCCTGAGCTCCGTGGTCACAGTGCCCTCTAGTTCACTGGGCACTCAGACCTATATCTGCAACGTGAATCACAAGCCAAGCAATACTAAAGTCGACAAGAAAGTGGAACCCAAGTCCTGTGATAAAACACATACTTGCCCACCTTGTCCTGCACCAGAGCTGCTGGGAGGACCATCCGTGTTCCTGTTTCCACCCAAGCCTAAAGACACTCTGATGATTTCTAGGACACCCGAAGTCACTTGCGTGGTCGTGGACGTGAGCCACGAGGACCCCGAAGTCAAGTTTAACTGGTACGTGGATGGCGTCGAGGTGCATAATGCTAAGACAAAACCTAGGGAGGAACAGTACAACAGTACATATAGAGTCGTGTCAGTCCTGACTGTGCTGCATCAGGACTGGCTGAACGGAAAGGAATATAAGTGCAAAGTGAGCAATAAGGCTCTGCCCGCACCTATCGAGAAAACTATTTCCAAGGCTAAAGGCCAGCCTAGAGAACCACAGGTGTACACCCTGCCTCCATCTAGGGACGAGCTGACTAAGAACCAGGTCAGTCTGACCTGTCTGGTGAAAGGCTTCTATCCTAGCGATATCGCAGTGGAGTGGGAATCCAATGGGCAGCCAGAGAACAATTACAAGACCACACCCCCTGTGCTGGACTCCGATGGGTCTTTCTTTCTGTATAGTAAGCTGACCGTCGATAAATCACGGTGGCAGCAGGGAAACGTGTTCAGCTGTAGTGTCATGCACGAAGCACTGCACAATCATTACACCCAGAAGAGCCTGTCACTGTCACCCGGATGA 15 Ramucirumab_LCGCCACCATGGCTGTGATGGCACCTAGAACACTGGTCCTGCTGCTGTCCGGGGCACTGGCACTGACTCAGACTTGGGCTGGCGATATTCAGATGACCCAGAGTCCAAGCTCCGTGTCCGCCTCTATCGGCGACCGAGTCACCATTACATGCAGAGCTAGCCAGGGCATCGATAACTGGCTGGGGTGGTACCAGCAGAAGCCTGGAAAAGCCCCAAAGCTGCTGATCTACGACGCTTCCAATCTGGATACAGGCGTGCCCTCTAGGTTCAGTGGCTCAGGGAGCGGAACTTACTTTACTCTGACCATCTCTAGTCTGCAGGCTGAGGACTTCGCAGTGTATTTTTGCCAGCAGGCAAAAGCCTTCCCCCCTACCTTTGGCGGGGGAACAAAAGTCGACATCAAGGGGACCGTGGCCGCTCCCTCAGTCTTCATTTTTCCACCCAGCGATGAGCAGCTGAAGTCTGGAACAGCCAGTGTGGTCTGTCTGCTGAACAATTTCTACCCTCGGGAAGCAAAAGTGCAGTGGAAGGTCGACAACGCCCTGCAGTCCGGCAACAGCCAGGAGAGTGTGACTGAACAGGACTCAAAAGATAGCACCTATTCCCTGTCAAGCACACTGACTCTGTCCAAGGCTGATTACGAAAAGCACAAAGTGTATGCATGTGAGGTCACCCATCAGGGGCTGTCAAGTCCAGTCACAAAAAGTTTCAACCGAGGAGAGTGCTGA 16 D3H44_HCGCCACAATGGCCGTGATGGCTCCTAGAACACTGGTCCTGCTGCTGTCCGGGGCTCTGGCTCTGACTCAGACTTGGGCTGGGGAGGTGCAGCTGGTCGAATCTGGAGGAGGACTGGTGCAGCCAGGAGGGTCACTGAGACTGAGCTGCGCCGCTTCCGGCTTCAACATCAAGGAGTACTATATGCACTGGGTGAGGCAGGCACCTGGCAAAGGACTGGAGTGGGTGGGACTGATCGACCCAGAACAGGGGAACACCATCTACGACCCTAAGTTTCAGGATAGGGCAACCATTTCTGCCGACAACAGTAAAAATACAGCTTATCTGCAGATGAACAGCCTGAGGGCTGAAGATACTGCAGTGTACTATTGCGCACGCGACACCGCAGCCTACTTCGATTATTGGGGACAGGGCACCCTGGTCACAGTGAGCTCCGCATCAACTAAGGGACCCAGCGTGTTTCCACTGGCCCCCTCTAGTAAATCCACTTCTGGAGGCACCGCTGCACTGGGCTGTCTGGTGAAGGATTACTTCCCAGAGCCCGTCACAGTGAGCTGGAACTCCGGGGCCCTGACCAGCGGAGTCCATACATTTCCTGCTGTGCTGCAGTCAAGCGGGCTGTACTCCCTGTCCTCTGTGGTCACCGTGCCAAGTTCAAGCCTGGGAACTCAGACCTATATCTGCAACGTGAATCACAAGCCTTCAAATACAAAAGTCGACAAGAAAGTGGAACCAAAGAGCTGTGATAAAACACATACTTGCCCACCTTGTCCTGCACCAGAGCTGCTGGGAGGACCAAGCGTGTTCCTGTTTCCACCCAAGCCCAAAGACACCCTGATGATTTCCCGCACACCAGAAGTCACTTGCGTGGTCGTGGACGTGTCTCACGAGGACCCCGAAGTCAAGTTCAACTGGTACGTGGATGGCGTCGAGGTGCATAATGCCAAGACAAAACCCCGGGAGGAACAGTACAACTCCACATATAGAGTCGTGTCTGTCCTGACTGTGCTGCACCAGGACTGGCTGAACGGGAAGGAGTATAAGTGCAAAGTGAGTAATAAGGCCCTGCCCGCTCCTATCGAGAAAACAATTAGCAAGGCCAAAGGCCAGCCTCGAGAACCACAGGTGTACACTCTGCCTCCATCTCGGGACGAGCTGACTAAGAACCAGGTCAGTCTGACCTGTCTGGTGAAAGGATTCTATCCCAGCGATATCGCTGTGGAGTGGGAATCCAATGGCCAGCCTGAGAACAATTACAAGACCACACCCCCTGTGCTGGACTCTGATGGCAGTTTCTTTCTGTATAGTAAGCTGACCGTCGATAAATCACGATGGCAGCAGGGGAACG GTTCAGCTGTTCAGTGATGCACGAAGCCCTGCACAACCATTACACCCAGAAGAGCCTGAGCCTGTCTCCCGGCTGA 17 D3H44_LCGCCACAATGGCTGTGATGGCACCCCGAACCCTGGTCCTGCTGCTGAGTGGAGCACTGGCACTGACCCAGACATGGGCAGGCGACATCCAGATGACACAGTCCCCTAGCTCCCTGAGTGCCTCAGTGGGGGACAGAGTCACTATCACCTGCCGGGCTTCCAGAGATATTAAGTCTTACCTGAACTGGTATCAGCAGAAGCCAGGCAAAGCACCCAAGGTGCTGATCTACTATGCCACCAGTCTGGCTGAAGGAGTGCCTTCACGGTTCAGCGGCTCCGGGTCTGGAACTGACTACACACTGACTATTTCTAGTCTGCAGCCTGAGGATTTCGCTACCTACTATTGCCTGCAGCACGGCGAATCCCCATGGACTTTTGGCCAGGGGACCAAAGTGGAGATCAAGAGGACAGTGGCCGCTCCATCCGTCTTCATTTTTCCCCCTTCTGACGAACAGCTGAAATCAGGAACTGCCAGCGTGGTCTGTCTGCTGAACAATTTCTACCCCCGCGAGGCAAAAGTGCAGTGGAAGGTCGATAACGCCCTGCAGAGTGGCAATTCACAGGAGAGCGTGACAGAACAGGACTCCAAAGATTCTACTTATAGTCTGTCAAGCACCCTGACACTGTCTAAGGCTGATTACGAGAAGCACAAAGTGTATGCATGCGAAGTCACCCATCAGGGGCTGTCCTCTCCCGTGACAAAGAGCTTTAATCGGGGAGAGTGTTGA 18 Pertuzumab_HCGCCACAATGGCTGTGATGGCTCCAAGAACCCTGGTCCTGCTGCTGTCCGGGGCTCTGGCTCTGACTCAGACCTGGGCCGGGGAAGTGCAGCTGGTCGAATCTGGAGGAGGACTGGTGCAGCCAGGAGGGTCCCTGCGCCTGTCTTGCGCCGCTAGTGGCTTCACTTTTACCGACTACACCATGGATTGGGTGCGACAGGCACCTGGAAAGGGCCTGGAGTGGGTCGCCGATGTGAACCCAAATAGCGGAGGCTCCATCTACAACCAGCGGTTCAAGGGCCGGTTCACCCTGTCAGTGGACCGGAGCAAAAACACCCTGTATCTGCAGATGAATAGCCTGCGAGCCGAAGATACTGCTGTGTACTATTGCGCCCGGAATCTGGGGCCCTCCTTCTACTTTGACTATTGGGGGCAGGGAACTCTGGTCACCGTGAGCTCCGCCTCCACCAAGGGACCTTCTGTGTTCCCACTGGCTCCCTCTAGTAAATCCACATCTGGGGGAACTGCAGCCCTGGGCTGTCTGGTGAAGGACTACTTCCCAGAGCCCGTCACAGTGTCTTGGAACAGTGGCGCTCTGACTTCTGGGGTCCACACCTTTCCTGCAGTGCTGCAGTCAAGCGGGCTGTACAGCCTGTCCTCTGTGGTCACCGTGCCAAGTTCAAGCCTGGGAACACAGACTTATATCTGCAACGTGAATCACAAGCCATCCAATACAAAAGTCGACAAGAAAGTGGAACCCAAGTCTTGTGATAAAACCCATACATGCCCCCCTTGTCCTGCACCAGAGCTGCTGGGAGGACCAAGCGTGTTCCTGTTTCCACCCAAGCCTAAAGATACACTGATGATTAGTAGGACCCCAGAAGTCACATGCGTGGTCGTGGACGTGAGCCACGAGGACCCCGAAGTCAAGTTTAACTGGTACGTGGACGGCGTCGAGGTGCATAATGCCAAGACTAAACCCAGGGAGGAACAGTACAACAGTACCTATCGCGTCGTGTCAGTCCTGACAGTGCTGCATCAGGATTGGCTGAACGGGAAAGAGTATAAGTGCAAAGTGAGCAATAAGGCTCTGCCCGCACCTATCGAGAAAACAATTTCCAAGGCAAAAGGACAGCCTAGAGAACCACAGGTGTACACTCTGCCTCCATCAAGGGATGAGCTGACAAAGAACCAGGTCAGCCTGACTTGTCTGGTGAAAGGATTCTATCCCTCTGACATTGCTGTGGAGTGGGAAAGTAATGGCCAGCCTGAGAACAATTACAAGACCACACCCCCTGTGCTGGACTCAGATGGCAGCTTCTTTCTGTATAGCAAGCTGACCGTCGACAAATCCCGGTGGCAGCAGGGGAATGTGTTTAGTTGTTCAGTCATGCACGAGGCACTGCACAACCATTACACCCAGAAGTCACTGTCACTGTCACCAGGGTGA 19 Pertuzumab_LCGCCACAATGGCTGTGATGGCACCTAGAACACTGGTCCTGCTGCTGAGCGGGGCACTGGCACTGACACAGACTTGGGCCGGGGATATTCAGATGACCCAGTCCCCAAGCTCCCTGAGTGCCTCAGTGGGCGACCGAGTCACCATCACATGCAAGGCTTCCCAGGATGTGTCTATTGGAGTCGCATGGTACCAGCAGAAGCCAGGCAAAGCACCCAAGCTGCTGATCTATAGCGCCTCCTACCGGTATACCGGCGTGCCCTCTAGATTCTCTGGCAGTGGGTCAGGAACAGACTTTACTCTGACCATCTCTAGTCTGCAGCCTGAGGATTTCGCTACCTACTATTGCCAGCAGTACTATATCTACCCATATACCTTTGGCCAGGGGACAAAAGTGGAGATCAAGAGGACTGTGGCCGCTCCCTCCGTCTTCATTTTTCCCCCTTCTGACGAACAGCTGAAAAGTGGCACAGCCAGCGTGGTCTGTCTGCTGAACAATTTCTACCCTCGCGAAGCCAAAGTGCAGTGGAAGGTCGATAACGCTCTGCAGAGCGGCAACAGCCAGGAGTCTGTGACTGAACAGGACAGTAAAGATTCAACCTATAGCCTGTCAAGCACACTGACTCTGAGCAAGGCAGACTACGAGAAGCACAAAGTGTATGCCTGCGAAGTCACACATCAGGGGCTGTCCTCTCCTGTGACTAAGAGCTTTAACAGAGGAGAGTGTTGA Note: Thenucleotide sequences start with the signal peptide sequence and end withthe stop codon TGA. These can be removed if desired.

TABLE A1 KABAT Heavy chain origin numbering D3H44 PERTUZUMAB TRASTUZUMABRAMUCIRUMAB  1 E E E E  2 V V V V  3 Q Q Q Q  4 L L L L  5 V V V V  6 EE E Q  7 S S S S  8 G G G G  9 G G G G  10 G G G G  11 L L L L  12 V V VV  13 Q Q Q K  14 P P P P  15 G G G G  16 G G G G  17 S S S S  18 L L LL  19 R R R R  20 L L L L  21 S S S S  22 C C C C  23 A A A A  24 A A AA  25 S S S S  26 G G G G  27 F F F F  28 N T N T  29 I F I F  32 K T KS  33 E D D S  34 Y Y T Y  35 Y T Y S   35A M M I M   35B H D H N  36 WW W W  37 V V V V  38 R R R R  39 Q Q Q Q  40 A A A A  41 P P P P  42 GG G G  43 K K K K  44 G G G G  45 L L L L  46 E E E E  47 W W W W  48 VV V V  49 G A A S  50 L D R S  51 I V I I  52 D N Y S   52A P P P S  52B E N T S  54 Q S N S  55 G G G S  56 N G Y Y  57 T S T I  58 I I RY  59 Y Y Y Y  60 D N A A  61 P Q D D  62 K R S S  63 F F V V  64 Q K KK  65 D G G G  66 R R R R  67 A F F F  68 T T T T  69 I L I I  70 S S SS  71 A V A R  72 D D D D  73 N R T N  74 S S S A  75 K K K K  76 N N NN  77 T T T S  78 A L A L  79 Y Y Y Y  80 L L L L  81 Q Q Q Q  82 M M MM   82A N N N N   82B S S S S   82C L L L L  83 R R R R  84 A A A A  85E E E E  86 D D D D  87 T T T T  88 A A A A  89 V V V V  90 Y Y Y Y  91Y Y Y Y  92 C C C C  93 A A S A  94 R R R R  95 D N W V  96 T L G T  97A G G D  98 P D G S F  98 A F Y  99 Y Y A A 100 F F M F 101 D D D D 102Y Y Y I 103 W W W W 104 G G G G 105 Q Q Q Q 106 G G G G 107 T T T T 108L L L M 109 V V V V 110 T T T T 111 V V V V 112 S S S S 113 S S S S 114A A A A 115 S S S S 116 T T T T 117 K K K K 118 G G G G 119 P P P P 120S S S S 121 V V V V 122 F F F F 123 P P P P 124 L L L L 125 A A A A 126P P P P 127 S S S S 128 S S S S 129 K K K K 130 S S S S 133 T T T T 134S S S S 135 G G G G 136 G G G G 137 T T T T 138 A A A A 139 A A A A 140L L L L 141 G G G G 142 C C C C 143 L L L L 144 V V V V 145 K K K K 146D D D D 147 Y Y Y Y 148 F F F F 149 P P P P 150 E E E E 151 P P P P 152V V V V 153 T T T T 154 V V V V 156 S S S S 157 W W W W 162 N N N N 163S S S S 164 G G G G 165 A A A A 166 L L L L 167 T T T T 168 S S S S 169G G G G 171 V V V V 172 H H H H 173 T T T T 174 F F F F 175 P P P P 176A A A A 177 V V V V 178 L L L L 179 Q Q Q Q 180 S S S S 182 S S S S 183G G G G 184 L L L L 185 Y Y Y Y 186 S S S S 187 L L L L 188 S S S S 189S S S S 190 V V V V 191 V V V V 192 T T T T 193 V V V V 194 P P P P 195S S S S 196 S S S S 197 S S S S 198 L L L L 199 G G G G 200 T T T T 203Q Q Q Q 205 T T T T 206 Y Y Y Y 207 I I I I 208 C C C C 209 N N N N 210V V V V 211 N N N N 212 H H H H 213 K K K K 214 P P P P 215 S S S S 216N N N N 217 T T T T 218 K K K K 219 V V V V 220 D D D D 221 K K K K 222K K K K 223 V V V V 226 E E E E 227 P P P P 228 K K K K 232 S S S S 233C C C C 234 D D D D 235 K K K K 236 T T T T 237 H H H H 238 T T T T 239C C C C 240 P P P P 241 P P P P 242 C C C C 243 P P P P 244 A A A A 245P P P P 246 E E E E 247 L L L L 248 L L L L 249 G G G G 250 G G G G 251P P P P 252 S S S S 253 V V V V 254 F F F F 255 L L L L 256 F F F F 257P P P P 258 P P P P 259 K K K K 260 P P P P 261 K K K K 262 D D D D 263T T T T 264 L L L L 265 M M M M 266 I I I I 267 S S S S 268 R R R R 269T T T T 270 P P P P 271 E E E E 272 V V V V 273 T T T T 274 C C C C 275V V V V 276 V V V V 277 V V V V 278 D D D D 279 V V V V 280 S S S S 281H H H H 282 E E E E 283 D D D D 284 P P P P 285 E E E E 286 V V V V 287K K K K 288 F F F F 289 N N N N 290 W W W W 291 Y Y Y Y 292 V V V V 295D D D D 296 G G G G 299 V V V V 300 E E E E 301 V V V V 302 H H H H 303N N N N 304 A A A A 305 K K K K 306 T T T T 307 K K K K 308 P P P P 309R R R R 310 E E E E 311 E E E E 312 Q Q Q Q 313 Y Y Y Y 314 N N N N 317S S S S 318 T T T T 319 Y Y Y Y 320 R R R R 321 V V V V 322 V V V V 323S S S S 324 V V V V 325 L L L L 326 T T T T 327 V V V V 328 L L L L 329H H H H 330 Q Q Q Q 331 D D D D 332 W W W W 333 L L L L 334 N N N N 335G G G G 336 K K K K 337 E E E E 338 Y Y Y Y 339 K K K K 340 C C C C 341K K K K 342 V V V V 343 S S S S 344 N N N N 345 K K K K 346 A A A A 347L L L L 348 P P P P 349 A A A A 350 P P P P 351 I I I I 352 E E E E 353K K K K 354 T T T T 355 I I I I 357 S S S S 358 K K K K 359 A A A A 360K K K K 361 G G G G 363 Q Q Q Q 364 P P P P 365 R R R R 366 E E E E 367P P P P 368 Q Q Q Q 369 V V V V 370 Y Y Y Y 371 T T T T 372 L L L L 373P P P P 374 P P P P 375 S S S S 376 R R R R 377 D D D D 378 E E E E 381L L L L 382 T T T T 383 K K K K 384 N N N N 385 Q Q Q Q 386 V V V V 387S S S S 388 L L L L 389 T T T T 390 C C C C 391 L L L L 392 V V V V 393K K K K 394 G G G G 395 F F F F 396 Y Y Y Y 397 P P P P 398 S S S S 399D D D D 400 I I I I 401 A A A A 402 V V V V 405 E E E E 406 W W W W 407E E E E 408 S S S S 410 N N N N 411 G G G G 414 Q Q Q Q 415 P P P P 416E E E E 417 N N N N 418 N N N N 419 Y Y Y Y 420 K K K K 421 T T T T 422T T T T 423 P P P P 424 P P P P 425 V V V V 426 L L L L 427 D D D D 428S S S S 430 D D D D 433 G G G G 434 S S S S 435 F F F F 436 F F F F 437L L L L 438 Y Y Y Y 439 S S S S 440 K K K K 441 L L L L 442 T T T T 443V V V V 444 D D D D 445 K K K K 446 S S S S 447 R R R R 448 W W W W 449Q Q Q Q 450 Q Q Q Q 451 G G G G 452 N N N N 453 V V V V 454 F F F F 455S S S S 456 C C C C 457 S S S S 458 V V V V 459 M M M M 460 H H H H 461E E E E 462 A A A A 463 L L L L 464 H H H H 465 N N N N 466 H H H H 467Y Y Y Y 468 T T T T 469 Q Q Q Q 470 K K K K 471 S S S S 472 L L L L 473S S S S 474 L L L L 475 S S S S 476 P P P P 477 G G G G Variableregions: HFR1; 1-30, CDR-H1; 31-35, HFR2; 36-49, CDR-H2; 50-65, HFR3;66-94, CDR-H3; 95-102, HFR4; 103-113 (Reference: Molecular Immunology.Volume 45, Issue 14, August 2008, Pages 3832-3839).

TABLE A2 KABAT Light chain origin numbering D3H44 PERTUZUMAB TRASTUZUMABRAMUCIRUMAB 1 D D D D 2 I I I I 3 Q Q Q Q 4 M M M M 5 T T T T 6 Q Q Q Q7 S S S S 8 P P P P 9 S S S S 10 S S S S 11 L L L V 12 S S S S 13 A A AA 14 S S S S 15 V V V I 16 G G G G 17 D D D D 18 R R R R 19 V V V V 20 TT T T 21 I I I I 22 T T T T 23 C C C C 24 R K R R 25 A A A A 26 S S S S27 R Q Q Q 28 D D D G 29 I V V I 30 K S N D 31 S I T N 32 Y G A W 33 L VV L 34 N A A G 35 W W W W 36 Y Y Y Y 37 Q Q Q Q 38 Q Q Q Q 39 K K K K 40P P P P 41 G G G G 42 K K K K 43 A A A A 44 P P P P 45 K K K K 46 V L LL 47 L L L L 48 I I I I 49 Y Y Y Y 50 Y S S D 51 A A A A 52 T S S S 53 SY F N 54 L R L L 55 A Y Y D 56 E T S T 57 G G G G 58 V V V V 59 P P P P60 S S S S 61 R R R R 62 F F F F 63 S S S S 64 G G G G 65 S S S S 66 G GR G 67 S S S S 68 G G G G 69 T T T T 70 D D D Y 71 Y F F F 72 T T T T 73L L L L 74 T T T T 75 I I I I 76 S S S S 77 S S S S 78 L L L L 79 Q Q QQ 80 P P P A 81 E E E E 82 D D D D 83 F F F F 84 A A A A 85 T T T V 86 YY Y Y 87 Y Y Y F 88 C C C C 89 L Q Q Q 90 Q Q Q Q 91 H Y H A 92 G Y Y K93 E I T A 94 S Y T F 95 P P P P 96 W Y P P 97 T T T T 98 F F F F 99 G GG G 100 Q Q Q G 101 G G G G 102 T T T T 103 K K K K 104 V V V V 105 E EE D 106 I I I I 107 K K K K 108 R R R G 109 T T T T 110 V V V V 111 A AA A 112 A A A A 113 P P P P 114 S S S S 115 V V V V 116 F F F F 117 I II I 118 F F F F 119 P P P P 120 P P P P 121 S S S S 122 D D D D 123 E EE E 124 Q Q Q Q 125 L L L L 126 K K K K 127 S S S S 128 G G G G 129 T TT T 130 A A A A 131 S S S S 132 V V V V 133 V V V V 134 C C C C 135 L LL L 136 L L L L 137 N N N N 138 N N N N 139 F F F F 140 Y Y Y Y 141 P PP P 142 R R R R 143 E E E E 144 A A A A 145 K K K K 146 V V V V 147 Q QQ Q 148 W W W W 149 K K K K 150 V V V V 151 D D D D 152 N N N N 153 A AA A 154 L L L L 155 Q Q Q Q 156 S S S S 157 G G G G 158 N N N N 159 S SS S 160 Q Q Q Q 161 E E E E 162 S S S S 163 V V V V 164 T T T T 165 E EE E 166 Q Q Q Q 167 D D D D 168 S S S S 169 K K K K 170 D D D D 171 S SS S 172 T T T T 173 Y Y Y Y 174 S S S S 175 L L L L 176 S S S S 177 S SS S 178 T T T T 179 L L L L 180 T T T T 181 L L L L 182 S S S S 183 K KK K 184 A A A A 185 D D D D 186 Y Y Y Y 187 E E E E 188 K K K K 189 H HH H 190 K K K K 191 V V V V 192 Y Y Y Y 193 A A A A 194 C C C C 195 E EE E 196 V V V V 197 T T T T 198 H H H H 199 Q Q Q Q 200 G G G G 201 L LL L 202 S S S S 203 S S S S 204 P P P P 205 V V V V 206 T T T T 207 K KK K 208 S S S S 209 F F F F 210 N N N N 211 R R R R 212 G G G G 213 E EE E 214 C C C C Variable regions: LFR1; 1-23, CDR-L1; 24-34, LFR2;35-49, CDR-L2; 50-56, LFR3; 57-88, CDR-L3; 89-97, LFR4; 98-110(Reference: Molecular Immunology. Volume 45, Issue 14, August 2008,Pages 3832-3839).

TABLE 14 Unique identifier set Buckets Fab Region Design TypeH1_mutation L1_mutation 1--2 Tm1 only, TF1 only constant electrostaticS186R Q124E_Q160E_T178D 3--4 constant steric F174V_P175S_S188G S176L5--6 constant electrostatic D146G_Q179K Q124E_Q160E_T180E 7--6 constantelectrostatic D146G_Q179R Q124E_Q160E_T180E 8--6 constant electrostaticD146G_S186R Q124E_Q160E_T180E 7--9 constant electrostatic D146G_Q179RQ124E_Q160E_T180E 8--9 constant electrostatic D146G_S186RQ124E_Q160E_T180E 10--11 constant steric F174V_S188L S176G 12--13constant steric F174V_P175S_S188G S176L 12--14 constant stericF174V_P175S_S188G S176L 12--15 constant steric F174V_P175S_S188G S176L16-17 variable electrostatic Q39D Q38R 18--11 constant stericF174W_S188L S176G 19--3  constant steric S188L_V190Y V133S 20--11constant steric S188L S176G 21-22 variable electrostatic Q39D Q38R 23-24constant electrostatic K145L_Q179E S131K 9--5 constant electrostaticK145T_Q179D_S188L Q160K_T178R 25-26 Tm1 only, TF1/TF2 variablecombination V37I_Q39R Q38E_F98W (electrostatic + steric) 27-28 variablecombination V37W_Q39E Q38R_F98A (electrostatic + steric) 29-30 variablesteric V37W_A93V F98A 31-32 variable steric WT F98W 33-34 variablecombination V37I_Q39R Q38E (electrostatic + steric) 35-36 Tm1 only,remaining variable combination Q39D Q38R TF category combinations(electrostatic + steric) 37-36 variable combination Q39E Q38R(electrostatic + steric) 25-38 variable combination V37I_Q39R Q38E_F98W(electrostatic + steric) 39-34 variable combination Q39R Q38E(electrostatic + steric) 39-40 variable combination Q39R Q38E(electrostatic + steric) 41-42 variable combination Q39D Q38R_F98W(electrostatic + steric) 43-17 variable electrostatic Q39E Q38R 22-44variable electrostatic Q39R Q38D 45-28 variable combination V37W_Q39DQ38R_F98A (electrostatic + steric) 46-30 variable steric V37W F98A 47-48variable electrostatic V37E_F100D L89R_F98W 49-42 variable combinationV37I_Q39E Q38R_F98W (electrostatic + steric) 50-42 variable combinationV37I_Q39D Q38R_F98W (electrostatic + steric) 51-52 variableelectrostatic Q39R Q38E 53-54 variable electrostatic Q39R Q38E 33-40variable combination V37I_Q39R Q38E (electrostatic + steric) 55-56variable electrostatic Q39M Q38M vs hydrophobic 57-58 Tm1/Tm2, TF1 onlyconstant electrostatic L143K_D146G Q124E_V133D  5-59 constantelectrostatic D146G_Q179K Q124E_Q160E_T180E  7-59 constant electrostaticD146G_Q179R Q124E_Q160E_T180E  8-59 constant electrostatic D146G_S186RQ124E_Q160E_T180E 60-61 constant electrostatic D146G_Q179RQ124E_Q160E_T178D 62-61 constant electrostatic D146G_S186RQ124E_Q160E_T178D 63-64 constant electrostatic D146G_S186RQ124E_Q160E_T178D 63-65 constant electrostatic D146G_S186RQ124E_Q160E_T178D 66-67 constant electrostatic L143A_D146G_Q179RQ124E_V133W_Q160E_T180E 66-68 constant electrostatic L143A_D146G_Q179RQ124E_V133W_Q160E_T180E 63-69 constant electrostatic D146G_S186RQ124E_Q160E_T178D  3-70 constant steric F174V_P175S_S188G S176L 61-71constant electrostatic L143E_K145T Q124R_Q160K_T178R 72-64 constantelectrostatic D146G_Q179R Q124E_Q160E_T178D 72-65 constant electrostaticD146G_Q179R Q124E_Q160E_T178D 72-69 constant electrostatic D146G_Q179RQ124E_Q160E_T178D 73-74 variable combination V37I_Q39R Q38E_F98W(electrostatic + steric) 75-76 variable electrostatic Q39D Q38R 75-77variable electrostatic Q39D Q38R 64-78 constant electrostaticK145E_D146G_Q179D_S188L Q160K_T178R 65-78 constant electrostaticK145T_Q179D_S188L Q160K_T178R 67-79 constant electrostaticK145T_Q179D_S188F V133A_Q160K_T178R 68-79 constant electrostaticK145T_Q179D_S188L V133A_Q160K_T178R 80-81 constant electrostaticK145T_Q179D_S188F Q160K_T178R 80-82 constant electrostaticK145T_Q179D_S188F Q160K_T178R 80-83 constant electrostaticK145T_Q179D_S188F Q160K_T178R 16-84 variable electrostatic Q39D Q38R85-81 constant electrostatic K145T_Q179D_S188L Q160K_T178R 85-82constant electrostatic K145T_Q179D_S188L Q160K_T178R 69-78 constantelectrostatic K145T_Q179D_S188F Q160K_T178R 85-83 constant electrostaticK145T_Q179D_S188L Q160K_T178R 86-87 variable electrostatic Q39KQ38N_T85E 86-88 variable electrostatic Q39K Q38N_T85E 89-90 Tm1/Tm2,TF1/TF2 variable combination Q39R Q38D_F98W (electrostatic + steric)73-91 variable combination V37I_Q39R Q38E_F98W (electrostatic + steric)92-90 variable combination V37I_Q39R Q38D_F98W (electrostatic + steric)93-26 variable combination Q39R Q38E_F98W (electrostatic + steric) 94-95variable combination V37I_Q39R Q38E_F98W (electrostatic + steric) 96-97Tm1/Tm2, remaining variable combination Q39D Q38R_F98W TF categorycombinations (electrostatic + steric) 89-98 variable combination Q39RQ38D_F98W (electrostatic + steric) 99-76 variable electrostatic Q39EQ38R 99-77 variable electrostatic Q39E Q38R 100-97  variable combinationV37I_Q39E Q38R_F98W (electrostatic + steric) 101-102 variableelectrostatic V37E L89R_F98T 103-104 constant steric A139G_V190AL135W_N137A 105-42  variable combination Q39E Q38R_F98W (electrostatic +steric) 106-97  variable combination V37I_Q39D Q38R_F98W(electrostatic + steric) 92-98 variable combination V37I_Q39R Q38D_F98W(electrostatic + steric) 43-84 variable electrostatic Q39E Q38R 93-38constant combination Q39R Q38E_F98W (electrostatic + steric) 107-108constant steric A139G_V190A L135W 109-110 variable electrostatic V37EL89R_F98V 111-112 Tm2 only, TF1 only combination combinationQ39D_A139G_V190A Q38R_L135W of constant (electrostatic + and variablesteric)  63-113 constant electrostatic D146G_S186R Q124E_Q160E_T178D 79-114 constant electrostatic D146G_Q179R Q124E_V133W_Q160E_T180E 66-114 constant electrostatic L143A_D146G_Q179R Q124E_V133W_Q160E_T180E 72-113 constant electrostatic D146G_Q179R Q124E_Q160E_T178D 113-78 constant electrostatic L143E_K145T Q160K_T178R 115-116 variablecombination Q39R Q38D_F98W (electrostatic + steric) 117-116 variablecombination V37I_Q39R Q38D_F98W (electrostatic + steric) 118-74 variable combination Q39R Q38E_F98W (electrostatic + steric) 119-120 Tm2only, TF1/TF2 variable combination Q39K Q38N_T85E_F98W (electrostatic +steric) 121-95  variable combination Q39R Q38E_F98W (electrostatic +steric) 115-122 variable combination Q39R Q38D_F98W (electrostatic +steric) 123-124 variable combination Q39R Q38D_F98W (electrostatic +steric) 117-122 variable combination V37I_Q39R Q38D_F98W(electrostatic + steric) 118-91  variable combination Q39R Q38E_F98W(electrostatic + steric) 125-124 variable combination V37I_Q39RQ38D_F98W (electrostatic + steric) 126-97  Tm2 only, remaining variablecombination Q39E Q38R_F98W TF category combinations (electrostatic +steric) 127-128 variable combination Q39E Q38N_T85K (electrostatic +steric) 129-128 variable combination V37I_Q39E Q38N_T85K(electrostatic + steric) 130-131 variable combination V37I_Q39EQ38N_T85K_F98W (electrostatic + steric)  96-132 Tm1/Tm3, any variablecombination Q39D Q38R_F98W TF catgory combination (electrostatic +steric)  3-133 constant steric F174V_P175S_S188G S176L 134-36  variablecombination V37I_Q39E Q38R (electrostatic + steric) 135-136 variablecombination Q39D Q38R (electrostatic + steric) 137-138 variablecombination Q39D Q38R_F98W (electrostatic + steric) 100-132 variablecombination V37I_Q39E Q38R_F98W (electrostatic + steric) 139-140constant combination A139I_K145T_D146G_Q179E_(—) F116A_V133G_S176F_T178A(electrostatic + S188G_V190S steric) 141-142 variable combination Q39EQ38R (electrostatic + steric)  73-143 variable combination V37I_Q39RQ38E_F98W (electrostatic + steric)  73-144 variable combinationV37I_Q39R Q38E_F98W (electrostatic + steric) 145-146 variablecombination Q39D Q38R_F98W (electrostatic + steric) 145-147 variablecombination Q39D Q38R_F98W (electrostatic + steric) 106-132 variablecombination V37I_Q39D Q38R_F98W (electrostatic + steric) 148-149variable combination V37A_Q39R_W103V Q38D_P44W (electrostatic + steric)148-150 variable combination V37A_Q39R_W103V Q38D_P44W (electrostatic +steric)  22-151 variable electrostatic Q39R Q38D 152-153 variablecombination V37A_Q39R_W103V Q38E_P44W (electrostatic + steric) 152-154variable combination V37A_Q39R_W103V Q38E_P44W (electrostatic + steric)155-36  variable combination V37I_Q39D Q38R (electrostatic + steric)156-157 variable steric V37T_A93Q_W103L P44W_F98W 158-159 variablecombination V37A_Q39R_W103V Q38E_P44W (electrostatic + steric) 160-146variable combination V37I_Q39E Q38R_F98W (electrostatic + steric)161-142 variable combination Q39D Q38R (electrostatic + steric) 160-147variable combination V37I_Q39E Q38R_F98W (electrostatic + steric)162-163 variable combination Q39D Q38R (electrostatic + steric) 164-165constant combination A139G_K145L_Q179E_V190A S131R_L135W(electrostatic + steric) 166-157 variable steric V37I_W103H P44W_F98W167-157 variable steric V37T_A93Q_W103V P44W_F98W 168-163 variablecombination Q39E Q38R (electrostatic + steric) 169-157 variable stericV37T_A93Q_W103T P44W_F98W 170-171 variable combination Q39K Q38N_T85E(electrostatic + steric) 170-172 variable combination Q39K Q38N_T85E(electrostatic + steric) 173-174 variable combination V37A_Q39R_W103VQ38D_P44W (electrostatic + steric)  94-175 variable combinationV37I_Q39R Q38E_F98W (electrostatic + steric) 157-176 variable stericV37W F98A 177-157 variable steric V37A_W103H P44W_F98W 157-178 variablesteric V37W F98A 179-180 variable steric V37W_F100W F98A 181-138variable combination V37I_Q39E Q38R_F98W (electrostatic + steric)182-183 variable electrostatic V37E_F100D L89R_F98W  86-184 variableelectrostatic Q39K Q38N_T85E 185-138 variable combination V37I_Q39DQ38R_F98W (electrostatic + steric) 186-187 variable steric WT F98W187-188 variable steric V37W_W103H F98L 189-157 variable stericV37A_W103V P44W_F98W 190-191 variable combination Q39E Q38R(electrostatic + steric) 192-193 Remaining categories combinationcombination Q39D_A139W Q38R_F116A_L135A of constant (electrostatic + andvariable steric) 194-195 combination combination A139G_V190A F98W_L135Wof constant (steric) and variable 196-197 combination combination A139WF98W_F116A_L135A of constant (steric) and variable 198-199 combinationcombination V37A_Q39E_W103H Q38N_P44W_T85K of constant (electrostatic +and variable steric) 200-146 variable combination Q39E Q38R_F98W(electrostatic + steric)  81-201 constant electrostatic D146G_Q179RQ124E_V133W_Q160E_T180E  82-201 constant electrostatic L143A_D146G_Q179RQ124E_V133W_Q160E_T180E 202-203 variable steric F100M_W103V P44W_L89W204-179 variable steric V37T_A93Q_W103V P44W_L89W_F98A 202-205 variablesteric F100M_W103V P44W_L89W 200-147 variable combination Q39E Q38R_F98W(electrostatic + steric) 206-136 variable combination Q39E Q38R(electrostatic + steric) 126-132 variable combination Q39E Q38R_F98W(electrostatic + steric) 207-203 variable steric F100M_W103H P44W_L89W207-205 variable steric F100M_W103H P44W_L89W 208-209 constantcombination A139G_S188G_V190A L135W_S176L_T178S (steric) 210-138variable combination Q39E Q38R_F98W (electrostatic + steric) 121-175variable combination Q39R Q38E_F98W (electrostatic + steric) 211-212constant combination A139S L135R (electrostatic + steric) 213-214variable combination V37A_Q39K_W103H Q38N_P44W_T85E (electrostatic +steric) 215-216 constant combination A139G_S188G_V190AV133G_L135W_S176F_T178A (steric) 115-217 variable combination Q39RQ38D_F98W (electrostatic + steric) 115-218 variable combination Q39RQ38D_F98W (electrostatic + steric) 123-219 variable combination Q39RQ38D_F98W (electrostatic + steric) 117-217 variable combinationV37I_Q39R Q38D_F98W (electrostatic + steric) 117-218 variablecombination V37I_Q39R Q38D_F98W (electrostatic + steric) 220-199variable combination V37A_Q39E_W103V Q38N_P44W_T85K (electrostatic +steric) 118-143 variable combination Q39R Q38E_F98W (electrostatic +steric) 118-144 variable combination Q39R Q38E_F98W (electrostatic +steric) 221-222 variable combination V37A_W103H_A139G_V190A P44W_L135W(steric) 223-179 variable steric V37T_A93Q_W103T P44W_L89W_F98A 224-225constant steric A139V_V190S WT 226-227 variable combination Q39KQ38N_T85E_F98W (electrostatic + steric) 226-228 variable combinationQ39K Q38N_T85E_F98W (electrostatic + steric) 229-230 variableelectrostatic V37E L89R_F98V 201-83  constant electrostaticK145E_D146G_Q179D_S188F Q160K_T178R 231-232 constant combinationA139I_K145T_D146G_S188G_(—) F116A_V133G_S176F_T178A (electrostatic +V190S steric) 233-131 variable combination Q39E Q38N_T85K_F98W(electrostatic + steric) 234-235 constant electrostaticK145E_D146G_Q179D_S188L T178R 236-237 constant combinationA139G_K145L_Q179E_V190A S131R_L135W (electrostatic + steric) 234-238constant electrostatic K145E_D146G_Q179D_S188L T178R 239-240 variablecombination Q39E Q38N_T85K_F98W (electrostatic + steric) 241-242variable steric V37W_W103H F98L 239-243 variable combination Q39EQ38N_T85K_F98W (electrostatic + steric) 242-244 variable stericV37A_W103H P44W 179-245 variable steric V37W_F100W F98A 246-225 constantsteric A139I_V190S WT 247-248 variable steric V37W_F100W F98A 125-219variable combination V37I_Q39R Q38D_F98W (electrostatic + steric)249-250 variable steric L45W Y87G 251-240 variable combination V37I_Q39EQ38N_T85K_F98W (electrostatic + steric) 252-227 variable combinationV37I_Q39K Q38N_T85E_F98W (electrostatic + steric) 251-243 variablecombination V37I_Q39E Q38N_T85K_F98W (electrostatic + steric) 252-228variable combination V37I_Q39K Q38N_T85E_F98W (electrostatic + steric)253-120 variable combination V37I_Q39K Q38N_T85E_F98W (electrostatic +steric) 254-255 constant steric F174G_S188A WT 250-256 variable stericV37A_W103H P44W Screening only/ Verification only/ Presence of H-LScreening and Unique disulfide verification data identifier bond (C233-for H1-L1:H1-L2 set H2_mutation L2_mutation C214) (y/n) (s/v/s_v)H1-L1:H1-L2 1--2 K145L_Q179E S131K n v 99:01 3--4 S188L V133S n s, v101:1_102:2_103:1_67:3_81:1_(—) 83:1_90:1_93:1_99:1 5--6K145E_D146G_Q179D_(—) Q160K_T178R n s, v 116:1_96:1 S188L 7--6K145E_D146G_Q179D_(—) Q160K_T178R n s, v 100:2_116:1 S188L 8--6K145E_D146G_Q179D_(—) Q160K_T178R n s, v 104:1_99:1 S188L 7--9K145T_Q179D_S188L Q160K_T178R n s, v 100:2_116:1 8--9 K145T_Q179D_S188LQ160K_T178R n s, v 104:1_99:1 10--11 F174V_P175S_S188G S176L n s, v91:10_92:10_84:15_107:19 12--13 F174V_S188L WT n s, v 53:10_67:11 12--14S188L WT n s, v 53:10_67:11 12--15 F174W_S188L WT n s, v 53:10_67:1116-17 V37I_Q39R Q38E_F98W n s, v 70:25_86:11 18--11 F174V_P175S_S188GS176L n s, v 71:26_79:15_87:23_94:13 19--3  F174V_P175S_S188G S176L n s,v 75:27_76:34_77:27_79:25_79:36_(—) 80:24_82:12_83:25_90:8 20--11F174V_P175S_S188G S176L n s, v 70:29_78:25_78:30 21-22 Q39R Q38D n s, v62:31_67:35 23-24 D146G_Q179K Q124E_Q160E_T180E n v 75:43 9--5D146G_Q179K Q124E_Q160E_T180E n s, v 54:43_57:31 25-26 V37W_Q39EQ38R_F98A n s, v 85:6_99:2 27-28 Q39R Q38D n s, v 71:18_87:9 29-30 WTL89F_F98W n s, v 64:17_88:7_89:18 31-32 V37W_A93V F98A n s, v101:20_17:83_62:27_67:29_67:29_(—) 78:17_82:18_83:18_83:21_83:23_(—)85:21_86:14_86:20 33-34 V37W_Q39E Q38R_F98A n s, v 56:34_74:24 35-36V37W_Q39R Q38E_F98A n s 102:01 37-36 V37W_Q39R Q38E_F98A n s, v103:1_82:8 25-38 V37W_Q39D Q38R_F98A n s, v 85:6_99:2 39-34 V37W_Q39EQ38R_F98A n s, v 104:1_60:18 39-40 V37W_Q39D Q38R_F98A n s, v104:1_60:18 41-42 V37W_Q39R Q38E_F98A n s, v 103:1_84:5_99:8 43-17V37I_Q39R Q38E_F98W n s, v 88:16_96:9 22-44 Q39E Q38R n s, v72:17_73:11_85:11 45-28 Q39R Q38D n s, v 101:16_80:15 46-30 WT L89F_F98Wn s, v 62:13_92:19_92:4 47-48 WT WT n s, v117:4_21:81_68:26_76:20_76:26_(—) 76:28_86:16_87:21_99:4 49-42 V37W_Q39RQ38E_F98A n s 81:24 50-42 V37W_Q39R Q38E_F98A n s, v 68:20_73:24 51-52Q39E Q38R n s, v 70:32_71:23_72:24_73:30_75:20_(—)76:24_78:24_81:15_85:8 53-54 WT WT n s, v65:38_66:19_69:22_73:38_74:29_(—) 74:31_77:27_77:36_78:30 33-40V37W_Q39D Q38R_F98A n s, v 56:34_74:24 55-56 Q39R Q38E n s, v59:41_60:31_70:33 57-58 L143E_K145T Q124R n s, v100:1_103:1_90:1_91:1_95:1_(—) 96:1_98:1_99:1  5-59 L143E_K145TQ160K_T178R n s, v 116:1_96:1  7-59 L143E_K145T Q160K_T178R n s, v100:2_116:1  8-59 L143E_K145T Q160K_T178R n s, v 104:1_99:1 60-61L143E_K145T Q124R_Q160K_T178R n s, v 102:1_105:1 62-61 L143E_K145TQ124R_Q160K_T178R n s, v 104:1_92:1 63-64 K145E_D146G_Q179D_(—)Q160K_T178R n s, v 112:1_116:1 S188L 63-65 K145T_Q179D_S188L Q160K_T178Rn s, v 112:1_116:1 66-67 K145T_Q179D_S188F V133A_Q160K_T178R n s, v101:1_104:1_107:1_111:2_112:1_(—) 115:1_94:1_97:1_99:1 66-68K145T_Q179D_S188L V133A_Q160K_T178R n s, v101:1_104:1_107:1_111:2_112:1_(—) 115:1_94:1_97:1_99:1 63-69K145T_Q179D_S188F Q160K_T178R n s, v 112:1_116:1  3-70 F174V_S188L V133Sn s, v 101:1_102:2_103:1_67:3_81:1_(—) 83:1_90:1_93:1_99:1 61-71D146G_Q179K Q124E_Q160E_T178D n s, v 77:1_88:3 72-64K145E_D146G_Q179D_(—) Q160K_T178R n s, v102:6_110:1_110:3_112:2_87:1_(—) S188L 88:5_94:4_95:3_99:1 72-65K145T_Q179D_S188L Q160K_T178R n s, v 102:6_110:1_110:3_112:2_87:1_(—)88:5_94:4_95:3_99:1 72-69 K145T_Q179D_S188F Q160K_T178R n s, v102:6_110:1_110:3_112:2_87:1_(—) 88:5_94:4_95:3_99:1 73-74V37W_Q39E_W103F Q38R_F98L n s, v 83:7_84:5 75-76 Q39R Q38D_F98W n s, v81:18_83:2 75-77 V37I_Q39R Q38D_F98W n s, v 81:18_83:2 64-78 D146G_Q179KQ124E_Q160E_T178D n s, v 84:15_91:4 65-78 D146G_Q179K Q124E_Q160E_T178Dn s, v 72:4_75:20_80:18_88:12_88:12_(—) 92:10_92:13_93:13_94:15 67-79D146G_Q179R Q124E_V133W_Q160E_(—) n s, v76:24_78:20_80:16_83:12_86:1_(—) T180E 86:21_88:13_93:11_94:17 68-79D146G_Q179R Q124E_V133W_Q160E_(—) n s, v 62:25_87:11 T180E 80-81D146G_Q179R Q124E_V133W_Q160E_(—) n s, v 72:18_77:15 T180E 80-82L143A_D146G_Q179R Q124E_V133W_Q160E_(—) n s, v 72:18_77:15 T180E 80-83D146G_Q179K Q124E_V133W_Q160E_(—) n s, v 72:18_77:15 T180E 16-84 Q39RQ38E_F98W n s, v 70:25_86:11 85-81 D146G_Q179R Q124E_V133W_Q160E_(—) ns, v 63:24_81:11 T180E 85-82 L143A_D146G_Q179R Q124E_V133W_Q160E_(—) ns, v 63:24_81:11 T180E 69-78 D146G_Q179K Q124E_Q160E_T178D n s, v76:18_82:19 85-83 D146G_Q179K Q124E_V133W_Q160E_(—) n s, v 63:24_81:11T180E 86-87 Q39E Q38N_T85K n s, v 55:30_66:22 86-88 V37I_Q39E Q38N_T85Kn s, v 55:30_66:22 89-90 V37W_Q39E Q38R_F98A n s, v 87:2_96:2 73-91V37W_Q39D_W103F Q38R_F98L n s, v 83:7_84:5 92-90 V37W_Q39E Q38R_F98A ns, v 82:6_91:11 93-26 V37W_Q39E Q38R_F98A n s, v71:24_75:13_75:19_76:3_79:12_(—) 82:12_83:6_89:6_97:1 94-95 V37W_W103FF98L n s, v 71:17_73:24 96-97 V37W_Q39R_W103F Q38E_F98L n s, v100:1_92:1 89-98 V37W_Q39D Q38R_F98A n s, v 87:2_96:2 99-76 Q39RQ38D_F98W n s, v 108:1_80:8 99-77 V37I_Q39R Q38D_F98W n s, v 108:1_80:8100-97  V37W_Q39R_W103F Q38E_F98L n s, v 85:10_85:2 101-102 WT WT n s, v101:6_101:8_107:4_54:25_86:8_(—) 91:14_91:17_97:6_98:5 103-104 A139WF116A_L135A n s 70:05 105-42  V37W_Q39R Q38E_F98A n s, v 74:15_90:3106-97  V37W_Q39R_W103F Q38E_F98L n s, v 92:15_97:6 92-98 V37W_Q39DQ38R_F98A n s, v 82:6_91:11 43-84 Q39R Q38E_F98W n s, v 88:16_96:9 93-38V37W_Q39D Q38R_F98A n s, v 71:24_75:13_75:19_76:3_79:12_(—)82:12_83:6_89:6_97:1 107-108 A139W F116A_L135A n s, v56:20_61:12_63:7_65:10_67:16_(—) 68:10_69:11_69:11_69:12_74:15 109-110WT WT n s, v 62:27_67:25_74:27 111-112 Q39R_A139W Q38D_F116A_L135A n s,v 111:1_85:1  63-113 L143E_K145T Q160K_T178R n s, v 112:1_116:1  79-114K145E_D146G_Q179D_(—) V133A_Q160K_T178R n s, v 108:1_93:2 S188F  66-114K145E_D146G_Q179D_(—) V133A_Q160K_T178R n s, v101:1_104:1_107:1_111:2_112:1_(—) S188F 115:1_94:1_97:1_99:1  72-113L143E_K145T Q160K_T178R n s, v 102:6_110:1_110:3_112:2_87:1_(—)88:5_94:4_95:3_99:1 113-78  D146G_Q179K Q124E_Q160E_T178D n s, v74:6_93:3_94:1 115-116 V37W_Q39E_W103F Q38R_F98L n s, v 55:16_89:3117-116 V37W_Q39E_W103F Q38R_F98L n s, v 85:8_86:13 118-74 V37W_Q39E_W103F Q38R_F98L n s, v 78:13_80:10_92:2 119-120 V37W_Q39EQ38N_T85K_F98A n s, v 56:9_69:1 121-95  V37W_W103F F98L n s, v71:15_93:3 115-122 V37W_Q39D_W103F Q38R_F98L n s, v 55:16_89:3 123-124V37W_W103F F98L n s, v 70:17_80:3 117-122 V37W_Q39D_W103F Q38R_F98L n s,v 85:8_86:13 118-91  V37W_Q39D_W103F Q38R_F98L n s, v 78:13_80:10_92:2125-124 V37W_W103F F98L n s, v 60:30_79:23 126-97  V37W_Q39R_W103FQ38E_F98L n s, v 101:1_82:16 127-128 V37W_Q39K Q38N_T85E_F98A n s, v101:2_86:13 129-128 V37W_Q39K Q38N_T85E_F98A n s, v 73:13_74:25 130-131V37W_Q39K Q38N_T85E_F98A n s, v 57:30_62:23  96-132 V37W_Q39R_W103HQ38E_F98L n s, v 100:1_92:1  3-133 F174W_S188L V133S n s, v101:1_102:2_103:1_67:3_81:1_(—) 83:1_90:1_93:1_99:1 134-36  V37W_Q39RQ38E_F98A n s 103:02 135-136 V37W_Q39R Q38D_F98A n s 104:01 137-138V37W_Q39R Q38D_F98A n s, v 100:1_93:7 100-132 V37W_Q39R_W103H Q38E_F98Ln s, v 85:10_85:2 139-140 S186K_S188H_V190G F118W_Q124E_V133S_(—) n s, v101:7_69:4 S176A_T178S_T180E 141-142 V37A_Q39R_W103V Q38D_P44W n s, v92:5_99:6  73-143 V37W_Q39D_W103H Q38R_F98L n s, v 83:7_84:5  73-144V37W_Q39E_W103H Q38R_F98L n s, v 83:7_84:5 145-146 V37W_Q39R_W103HQ38D_F98L n s, v 36:73_86:3_87:8 145-147 V37W_Q39R_W103F Q38D_F98L n s,v 36:73_86:3_87:8 106-132 V37W_Q39R_W103H Q38E_F98L n s, v 92:15_97:6148-149 V37W_Q39D Q38R_F98A n s, v 68:6_69:9 148-150 V37W_Q39E Q38R_F98An s, v 68:6_69:9  22-151 V37I_Q39R Q38R n s, v 72:17_73:11_85:11 152-153V37W_Q39D Q38R_F98A n s, v 61:19_63:5 152-154 V37W_Q39E Q38R_F98A n s, v61:19_63:5 155-36  V37W_Q39R Q38E_F98A n s, v 58:32_94:6 156-157 V37WF98A n s, v 50:17_76:8 158-159 V37W F98A n s, v 66:13_77:15 160-146V37W_Q39R_W103H Q38D_F98L n s 88:18 161-142 V37A_Q39R_W103V Q38D_P44W ns, v 102:8_56:29 160-147 V37W_Q39R_W103F Q38D_F98L n s 88:18 162-163V37A_Q39R_W103V Q38E_P44W n s, v 77:23_98:16 164-165 A139W F116A_L135A ns, v 48:21_49:14_50:12_51:10_68:1 166-157 V37W F98A n s, v 55:20_61:10167-157 V37W F98A n s, v 50:21_74:10 168-163 V37A_Q39R_W103V Q38E_P44W ns, v 60:32_91:10 169-157 V37W F98A n s, v 54:26_68:9 170-171V37A_Q39E_W103H Q38N_P44W_T85K n s, v 66:34_92:13 170-172V37A_Q39E_W103V Q38N_P44W_T85K n s, v 66:34_92:13 173-174 V37W F98A n s,v 61:12_74:26  94-175 V37W_W103H F98L n s, v 71:17_73:24 157-176 W103HP44W_F98W n s, v 63:37_86:12 177-157 V37W F98A n s 66:19 157-178 W103VP44W_F98W n s, v 63:37_86:12 179-180 W103V P44W_L89W_F98A n s, v64:26_69:26_69:28_75:25_77:26_(—) 78:26_83:22_86:16_95:8 181-138V37W_Q39R Q38D_F98A n s 84:29 182-183 V37S_A93K F98Y n s, v65:34_68:24_75:27_77:18_77:18_(—) 79:31_81:28_86:21_91:16  86-184 Q39DQ38N_T85K n s, v 55:30_66:22 185-138 V37W_Q39R Q38D_F98A n s 67:31186-187 V37W_W103H F98L n s, v 50:36_51:24_74:28 187-188 V37I F98W n s,v 57:30_62:31 189-157 V37W F98A n s 69:39 190-191 V37W F98A n s, v48:30_57:32_75:7 192-193 Q39R_A139G_V190A Q38D_L135W n s 103:01 194-195V37W_W103H_A139W F98L_F116A_L135A n s 81:01 196-197 V37W_W103H_A139G_(—)F98L_L135W n s 71:01 V190A 198-199 V37W_Q39K Q38N_T85E_F98A n s, v67:1_76:1 200-146 V37W_Q39R_W103H Q38D_F98L n s, v 102:2_96:1  81-201K145E_D146G_Q179D_(—) Q160K_T178R n s, v 102:1_113:3 S188F  82-201K145E_D146G_Q179D_(—) Q160K_T178R n s, v 103:1_97:3 S188F 202-203V37W_F100W F98A n s, v 86:5_87:2_97:1 204-179 V37W_F100W F98A n s, v87:2_88:1 202-205 V37W_F100W_W103L F98A n s, v 86:5_87:2_97:1 200-147V37W_Q39R_W103F Q38D_F98L n s, v 102:2_96:1 206-136 V37W_Q39R Q38D_F98An s, v 104:2_93:1 126-132 V37W_Q39R_W103H Q38E_F98L n s, v 101:1_82:16207-203 V37W_F100W F98A n s, v 92:5_93:6_98:1 207-205 V37W_F100W_W103LF98A n s, v 92:5_93:6_98:1 208-209 A139W_S188H_V190S F116S_L135A_S176A ns 85:06 210-138 V37W_Q39R Q38D_F98A n s, v 100:4_74:8 121-175 V37W_W103HF98L n s, v 71:15_93:3 211-212 A139I F118W_V133S n s 60:05 213-214V37W_Q39E Q38N_T85K_F98A n s, v 60:11_76:3 215-216 A139W_S188AF116S_L135A_S176A n s 84:07 115-217 V37W_Q39D_W103H Q38R_F98L n s, v55:16_89:3 115-218 V37W_Q39E_W103H Q38R_F98L n s, v 55:16_89:3 123-219V37W_W103H F98L n s, v 70:17_80:3 117-217 V37W_Q39D_W103H Q38R_F98L n s,v 85:8_86:13 117-218 V37W_Q39E_W103H Q38R_F98L n s, v 85:8_86:13 220-199V37W_Q39K Q38N_T85E_F98A n s, v 72:12_91:7 118-143 V37W_Q39D_W103HQ38R_F98L n s, v 78:13_80:10_92:2 118-144 V37W_Q39E_W103H Q38R_F98L n s,v 78:13_80:10_92:2 221-222 V37W_A139W F98A_F116A_L135A y s 68:10 223-179V37W_F100W F98A n s 85:12 224-225 A139I F118W_V133S n s, v52:9_57:9_59:35 226-227 V37W_Q39E_W103H Q38N_T85K_F98L n s, v37:27_67:12_80:9 226-228 V37W_Q39E_W103F Q38N_T85K_F98L n s, v37:27_67:12_80:9 229-230 V37S_A93K F98Y n s, v 110:16_76:21 201-83 D146G_Q179K Q124E_V133W_Q160E_(—) n s, v 69:21_86:10 T180E 231-232S186K_S188H_V190G F118W_V133S_S176A_(—) n s 69:16 T178S_T180E 233-131V37W_Q39K Q38N_T85E_F98A n s, v 49:28_88:10 234-235 D146G_S186RQ124E_Q160E_T178D n s, v 67:25_78:15 236-237 A139W F116S_L135A n s 54:14234-238 D146G_Q179R Q124E_Q160E_T178D n s, v 67:25_78:15 239-240V37W_Q39K_W103H Q38N_T85E_F98L n s, v 72:29_86:15 241-242 V37A_W103HP44W n s, v 48:29_54:22_56:21_75:10_81:14_(—) 88:9 239-243V37W_Q39K_W103F Q38N_T85E_F98L n s, v 72:29_86:15 242-244V37T_A93Q_W103T F98L n s, v 36:25_41:25_58:7_67:6 179-245 W103HP44W_L89W_F98A n s, v 64:26_69:26_69:28_75:25_77:26_(—)78:26_83:22_86:16_95:8 246-225 A139I F118W_V133S n s, v 67:28_69:24_77:8247-248 V37W_F100W_W103L L89W_F98A n s, v 67:30_71:22 125-219 V37W_W103HF98L n s, v 60:30_79:23 249-250 V37A_W103H P44W n s, v 55:24_58:31251-240 V37W_Q39K_W103H Q38N_T85E_F98L n s, v 62:38_64:27 252-227V37W_Q39E_W103H Q38N_T85K_F98L n s 68:35 251-243 V37W_Q39K_W103FQ38N_T85E_F98L n s, v 62:38_64:27 252-228 V37W_Q39E_W103F Q38N_T85K_F98Ln s 68:35 253-120 V37W_Q39E Q38N_T85K_F98A n s 67:36 254-255 F174A_S188GS176W n s, v 73:30_78:30_66:32_61:36_65:37_(—) 70:38_59:40_62:40_54:45250-256 V37T_A93Q_W103T Y87G n s, v 43:26_43:27_53:19 Screeningonly/Verifi- cation only/ Screening H1-L1 H2-L2 and verifi- H1-L1 H2-L2Antigen Antigen Unique Normal- cation data Normal- Tm Tm AffinityAffinity identi- ized Me- for H2- ized Me- (Range (Range (KD) (Range(KD) (Range fier dian H1- L2:H2-L1 dian H2- if n > 1) if n > 1) ifn > 1) if n > 1) set L1:H1-L2 (s/v/s_v) H2-L2:H2-L1 L2:H2-L1 (° C.) (°C.) (nM) (nM) 1--2 99:01 v 100:06 95:05 72.52 74.38 0.125 0.066 3--499:01 s, v 73:10_80:14 87:13 73.3 73.22 0.0399 0.0144 5--6 99:01 s, v61:30_64:32 67:33 74.32 74.95 0.0245 0.0415 7--6 99:01 s, v 61:30_64:3267:33 71.85 74.95 0.078 0.0415 8--6 99:01 s, v 61:30_64:32 67:33 72.5774.95 0.0671 0.0415 7--9 99:01 s, v 54:43_57:31 60:40 71.85 76.31 0.0780.0705 8--9 99:01 s, v 54:43_57:31 60:40 72.57 76.31 0.0671 0.070510--11 88:12 s 85:30 74:26 73.88 73.3 0.0949 0.0399 12--13 86:14 s, v103:6_76:23_92:22_94:6 89:11 73.3 71.62 0.0399 0.0377 (0.0386) 12--1486:14 s, v 60:37_83:10_92:9_97:33 83:17 73.3 76.91 0.0399 0.0342(0.0372) 12--15 86:14 s, v 76:21_78:18 80:20 73.3 73.01 0.0399 0.027116-17 82:18 s, v 58:25_59:45 64:36 73.03 72.38 0.077 0.144 18--11 81:19s 85:30 74:26 72.21 73.3 0.0155 0.0399 19--3  76:24 s, v101:1_102:2_103:1_67:3_81:1_(—) 99:01 74.73 73.3 0.0428 0.039983:1_90:1_93:1_99:1 20--11 72:28 s 85:30 74:26 77.09 73.3 0.0866 0.039921-22 66:34 s, v 72:17_73:11_85:11 87:13 73.03 71.68 0.077 0.0229 23-2464:36 v 126:01 99:01 74.38 74.32 0.066 0.0245 9--5 60:40 s, v 116:1_96:199:01 76.31 74.32 0.0705 0.0245 25-26 96:04 s, v101:4_71:18_73:17_85:12_85:18_(—) 90:10 72.38 73.07 0.144 0.90286:9_88:7_89:10_99:2 27-28 86:14 s 99:02 98:02 73.07 71.68 0.902 0.022929-30 83:17 s, v 31:60_56:31_82:25_97:17 71:29 76.47 74.7 0.697 0.23931-32 80:20 s, v 37:57_56:19_72:39_83:18 70:30 74.74 76.47 0.0109 0.69733-34 69:31 s, v 67:13_90:8 88:12 73.6 73.07 0.123 0.902 35-36 99:01 s,v 62:26_62:29 69:31 73.64 71.29 0.0524 3.18 37-36 97:03 s, v 62:26_62:2969:31 73.02 71.29 ND 3.18 (0.22) 25-38 96:04 s, v 107:10_80:11 90:1072.38 73.21 0.144 1.73 39-34 95:05 s, v 67:13_90:8 88:12 72.78 73.07 ND0.902 (0.006) 39-40 95:05 s, v 86:15_91:27 82:18 72.78 73.21 ND 1.73(0.006) 41-42 95:05 s, v 57:18_65:24 75:25 71.9 71.29 0.127 3.18 43-1789:11 s, v 58:25_59:45 64:36 73.02 72.38 ND 0.144 (0.22) 22-44 87:13 s60:18 77:23 71.68 73.02 0.0229 ND (0.22) 45-28 85:15 s 99:02 98:02 73.2171.68 1.73 0.0229 46-30 83:17 s, v 31:60_56:31_82:25_97:17 71:29 76.974.7 3.44 0.239 47-48 80:20 s, v 76:25_78:27_83:14_85:18_85:6_(—) 85:1575.84 75.83 NB 0.056 86:14_90:10_91:16 (0.7) (2.1884) (0.1017) 49-4277:23 s, v 57:18_65:24 75:25 72.87 71.29 0.0729 3.18 50-42 76:24 s, v57:18_65:24 75:25 73.78 71.29 0.177 3.18 51-52 76:24 s, v63:30_65:30_71:31_76:35_77:31_(—) 70:30 72.78 73.02 ND ND81:35_91:26_91:27_97:15 (0.006) (0.22) 53-54 72:28 s, v58:37_59:33_60:45_74:34_77:32_(—) 70:30 72.78 75.83 ND 0.05679:30_81:34_82:34 (0.006) (2.1884) (0.1017) 33-40 69:31 s, v 86:15_91:2782:18 73.6 73.21 0.123 1.73 55-56 66:34 s, v 68:24_71:27_83:17 74:2672.58 72.78 0.0273 ND (0.06) 57-58 99:01 s, v106:1_107:1_107:2_112:1_112:1_(—) 99:01 67.04 75.45 0.0243 0.0596117:2_120:1_123:1_92:2  5-59 99:01 s, v 73:5_83:1_83:2 98:02 74.32 69.60.0245 0.0236  7-59 99:01 s, v 73:5_83:1_83:2 98:02 71.85 69.6 0.0780.0236  8-59 99:01 s, v 73:5_83:1_83:2 98:02 72.57 69.6 0.0671 0.023660-61 99:01 s, v 77:1_88:3 98:02 70.11 72.7 0.0816 0.0769 62-61 99:01 s,v 77:1_88:3 98:02 66.67 72.7 0.0511 0.0769 63-64 99:01 s, v 84:15_91:492:08 66.67 74.95 0.0511 0.0415 63-65 99:01 s, v72:4_75:20_80:18_88:12_88:12_(—) 88:12 66.67 76.31 0.0511 0.070592:10_92:13_93:13_94:15 66-67 99:01 s, v76:24_78:20_80:16_83:12_86:1_(—) 85:15 67.53 71.45 0.0871 0.085686:21_88:13_93:11_94:17 66-68 99:01 s, v 62:25_87:11 82:18 67.53 73.140.0871 0.0423 63-69 99:01 s, v 76:18_82:19 81:19 66.67 74.3 0.0511 0.111 3-70 99:01 s, v 70:24_80:28 74:26 73.3 66.245 0.0399 0.0201 (0.99)61-71 98:02 v 102:01 99:01 72.7 69.16 0.0769 0.0525 72-64 98:02 s, v84:15_91:4 92:08 70.11 74.95 0.0816 0.0415 72-65 98:02 s, v72:4_75:20_80:18_88:12_88:12_(—) 88:12 70.11 76.31 0.0816 0.070592:10_92:13_93:13_94:15 72-69 98:02 s, v 76:18_82:19 81:19 70.11 74.30.0816 0.111 73-74 93:07 s, v 79:10_86:8 90:10 72.38 66.23 0.144 0.17275-76 93:07 s, v 72:13_83:11 87:13 73.03 67.79 0.077 0.127 75-77 93:07s, v 59:41_67:32 63:37 73.03 69.62 0.077 0.101 64-78 92:08 v 81:03 97:0374.95 69.16 0.0415 0.0525 65-78 88:12 v 81:03 97:03 76.31 69.16 0.07050.0525 67-79 85:15 s, v 108:1_93:2 99:01 71.45 67.01 0.0856 0.0631 68-7982:18 s, v 108:1_93:2 99:01 73.14 67.01 0.0423 0.0631 80-81 82:18 s, v102:1_113:3 98:02 74.3 67.01 0.111 0.0631 80-82 82:18 s, v 103:1_97:398:02 74.3 67.53 0.111 0.0871 80-83 82:18 v 84:03 96:04 74.3 68.37 0.1110.0828 16-84 82:18 s, v 80:17_85:12 85:15 73.03 70.69 0.077 0.1785(0.007) 85-81 81:19 s, v 102:1_113:3 98:02 76.31 67.01 0.0705 0.063185-82 81:19 s, v 103:1_97:3 98:02 76.31 67.53 0.0705 0.0871 69-78 81:19v 81:03 97:03 74.3 69.16 0.111 0.0525 85-83 81:19 v 84:03 96:04 76.3168.37 0.0705 0.0828 86-87 70:30 s, v 59:33_72:30 67:33 71.25 68.830.0213 0.0201 86-88 70:30 s 73:37 66:34 71.25 69.8 0.0213 0.0182 89-9098:02 s, v 74:12_93:4 92:08 67.79 73.07 0.127 0.902 73-91 93:07 s, v115:11_99:4 94:06 72.38 67.39 0.144 0.924 92-90 91:09 s, v 74:12_93:492:08 69.62 73.07 0.101 0.902 93-26 87:13 s, v101:4_71:18_73:17_85:12_85:18_(—) 90:10 70.69 73.07 0.1785 0.90286:9_88:7_89:10_99:2 (0.007) 94-95 78:22 s, v 61:12_83:13 85:15 72.3869.42 0.144 0.955 (1.05) 96-97 99:01 s, v 72:10_99:2 95:05 71.9 67.140.127 1.11 89-98 98:02 s, v 90:8_93:7 92:08 67.79 73.21 0.127 1.73 99-7697:03 s, v 72:13_83:11 87:13 73.02 67.79 ND 0.127 (0.22) 99-77 97:03 s,v 59:41_67:32 63:37 73.02 69.62 ND 0.101 (0.22) 100-97  95:05 s, v72:10_99:2 95:05 72.87 67.14 0.0729 1.11 101-102 93:07 s, v75:28_78:2_81:23_84:21_85:23_(—) 83:17 68 75.83 2.41 0.05686:13_86:18_88:5_91:16 (2.1884) (0.1017) 103-104 93:07 s 74:20 79:2170.65 71.86 ND ND 105-42  92:08 s, v 57:18_65:24 75:25 70.74 71.29 0.1633.18 106-97  91:09 s, v 72:10_99:2 95:05 73.78 67.14 0.177 1.11 92-9891:09 s, v 90:8_93:7 92:08 69.62 73.21 0.101 1.73 43-84 89:11 s, v80:17_85:12 85:15 73.02 70.69 ND 0.1785 (0.22) (0.007) 93-38 87:13 s, v107:10_80:11 90:10 70.69 73.21 0.1785 1.73 (0.007) 107-108 86:14 s, v100:11_77:17_79:10_90:17_93:11_(—) 89:11 70.88 71.86 0.0586 ND96:10_96:15_96:6_99:12 (0.32) 109-110 73:27 s, v 70:3_74:29 88:12 69.87575.83 NB 0.056 (0.03) (2.1884) (0.1017) 111-112 99:01 s, v 101:1_84:299:01 69.22 66.73 0.117 5.64E−09  63-113 99:01 s, v 74:6_93:3_94:1 97:0366.67 69.6 0.0511 0.0236  79-114 99:01 s, v 66:24_93:11 83:17 67.0168.64 0.0631 0.0219  66-114 99:01 s, v 66:24_93:11 83:17 67.53 68.640.0871 0.0219  72-113 98:02 s, v 74:6_93:3_94:1 97:03 70.11 69.6 0.08160.0236 113-78  97:03 v 81:03 97:03 69.6 69.16 0.0236 0.0525 115-11691:09 s, v 75:8_89:8 91:09 67.79 66.23 0.127 0.172 117-116 90:10 s, v75:8_89:8 91:09 69.62 66.23 0.101 0.172 118-74  89:11 s, v 79:10_86:890:10 70.69 66.23 0.1785 0.172 (0.007) 119-120 95:05 s, v 61:35_74:1972:28 66.94 67.29 0.0134 0.861 121-95  93:07 s, v 61:12_83:13 85:1570.69 69.42 0.1785 0.955 (0.007) (1.05) 115-122 91:09 s, v 114:5_91:297:03 67.79 67.39 0.127 0.924 123-124 91:09 s, v 75:8_85:8 91:09 67.7969.42 0.127 0.955 (1.05) 117-122 90:10 s, v 114:5_91:2 97:03 69.62 67.390.101 0.924 118-91  89:11 s, v 115:11_99:4 94:06 70.69 67.39 0.17850.924 (0.007) 125-124 72:28 s, v 75:8_85:8 91:09 69.62 69.42 0.101 0.955(1.05) 126-97  96:04 s, v 72:10_99:2 95:05 70.74 67.14 0.163 1.11127-128 95:05 s, v 64:29_70:21 73:27 68.83 68.63 0.0201 1.78 129-12880:20 s, v 64:29_70:21 73:27 69.8 68.63 0.0182 1.78 130-131 69:31 s, v81:16_88:10 87:13 68.1 68.63 0.1247 1.78 (0.0646)  96-132 99:01 s, v76:6_95:3 95:05 71.9 58.03 0.127 2.99  3-133 99:01 s, v 68:21_72:2476:24 73.3 65.53 0.0399 0.0172 134-36  99:01 s, v 62:26_62:29 69:31 ND71.29 ND 3.18 135-136 99:01 s, v 47:31_53:39_93:38 60:40 73.64 ND 0.0524ND 137-138 97:03 s, v 46:21_46:34_97:20 69:31 71.9 ND 0.127 ND 100-13295:05 s, v 76:6_95:3 95:05 72.87 58.03 0.0729 2.99 139-140 94:06 s, v90:1_98:5 98:02 65 72.57 0.0406 0.0615 141-142 94:06 s, v 61:18_69:1679:21 73.02 ND ND ND (0.22)  73-143 93:07 s, v 94:1_97:1 99:01 72.3858.76 0.144 7.89  73-144 93:07 s, v 105:3_57:5_97:1 97:03 72.38 ND 0.144ND 145-146 92:08 s, v 83:6_98:3 95:05 71.9 60.35 0.127 5.62 145-14792:08 s, v 48:24_63:33 66:34 71.9 ND 0.127 ND 106-132 91:09 s, v76:6_95:3 95:05 73.78 58.03 0.177 2.99 148-149 90:10 s, v 101:3_99:397:03 ND 73.21 ND 1.73 148-150 90:10 s, v 74:8_84:4 93:07 ND 73.07 ND0.902  22-151 87:13 s 54:34 61:39 71.68 ND 0.0229 ND 152-153 86:14 s, v101:5_116:4 96:04 60.29 73.21 0.977 1.73 152-154 86:14 s, v 86:12_91:393:07 60.29 73.07 0.977 0.902 155-36  85:15 s, v 62:26_62:29 69:31 ND71.29 ND 3.18 156-157 84:16 s, v 63:37_86:12 77:23 53.23 76.9 NB 3.44158-159 84:16 s, v 58:38_87:31 68:32 60.29 76.9 0.977 3.44 160-146 83:17s, v 83:6_98:3 95:05 72.87 60.35 0.0729 5.62 161-142 83:17 s, v61:18_69:16 79:21 73.03 ND 0.077 ND 160-147 83:17 s, v 48:24_63:33 66:3472.87 ND 0.0729 ND 162-163 82:18 s, v 42:33_67:12_69:8 85:15 73.03 60.290.077 0.977 164-165 81:19 s, v 104:2_105:1_107:1_108:1_110:1_(—) 99:01ND 71.86 ND ND 111:1_115:1_117:1_120:1 166-157 81:19 s, v 63:37_86:1277:23 59.34 76.9 0.24 3.44 167-157 81:19 s, v 63:37_86:12 77:23 54.8976.9 0.192 3.44 168-163 80:20 s, v 42:33_67:12_69:8 85:15 73.02 60.29 ND0.977 (0.22) 169-157 80:20 s, v 63:37_86:12 77:23 53.64 76.9 1.33 3.44170-171 79:21 s, v 63:3_75:7 94:06 71.25 55.37 0.0213 NB 170-172 79:21s, v 60:17_70:17 79:21 71.25 59.74 0.0213 0.55 173-174 79:21 s, v101:28_61:40 70:30 ND 76.9 ND 3.44  94-175 78:22 s, v 79:12_85:7 90:1072.38 60.9 0.144 ND 157-176 77:23 s, v 65:6_66:12 88:12 76.9 58.86 3.440.212 177-157 77:23 s, v 63:37_86:12 77:23 ND 76.9 ND 3.44 157-178 77:23s, v 52:18_64:21 75:25 76.9 63.59 3.44 0.174 179-180 75:25 s, v102:4_104:3_105:2_81:3_92:3_(—) 97:03 27.52 74.17 NB 4.12596:1_96:3_99:2_99:3 (0.35) 181-138 75:25 s, v 46:21_46:34_97:20 69:3172.87 ND 0.0729 ND 182-183 74:26 s, v 66:12_69:15_69:7_72:1_72:11_(—)89:11 75.84 61.7 NB 0.42 75:12_76:9_80:8_82:7 (0.7)  86-184 70:30 s72:27 72:28 71.25 ND 0.0213 ND 185-138 69:31 s, v 46:21_46:34_97:2069:31 73.78 ND 0.177 ND 186-187 68:32 s, v 57:30_62:31 66:34 74.74 60.90.0109 ND 187-188 66:34 s 66:22 75:25 60.9 75.745 ND ND (0.11) 189-15764:36 s, v 63:37_86:12 77:23 ND 76.9 ND 3.44 190-191 64:36 s, v48:27_49:27_53:33 64:36 ND 76.9 ND 3.44 192-193 99:01 s 88:01 99:01 NDND ND ND 194-195 99:01 s 69:01 99:01 ND ND ND ND 196-197 99:01 s 66:0396:04 ND ND ND ND 198-199 99:01 s, v 68:15_74:21 80:20 55.37 68.63 NB1.78 200-146 98:02 s, v 83:6_98:3 95:05 70.74 60.35 0.163 5.62  81-20198:02 s, v 69:21_86:10 84:16 67.01 ND 0.0631 ND  82-201 98:02 s, v69:21_86:10 84:16 67.53 ND 0.0871 ND 202-203 98:02 s, v 79:28_80:1480:20 70.92 27.52 1.54 NB 204-179 98:02 s, v64:26_69:26_69:28_75:25_77:26_(—) 75:25 64.69 27.52 NB NB78:26_83:22_86:16_95:8 202-205 98:02 s, v 60:33_77:17 74:26 70.92 64.561.54 1.22 200-147 98:02 s, v 48:24_63:33 66:34 70.74 ND 0.163 ND 206-13698:02 s, v 47:31_53:39_93:38 60:40 ND ND ND ND 126-132 96:04 s, v76:6_95:3 95:05 70.74 58.03 0.163 2.99 207-203 95:05 s, v 79:28_80:1480:20 ND 27.52 ND NB 207-205 95:05 s, v 60:33_77:17 74:26 ND 64.56 ND1.22 208-209 94:06 s 89:03 97:03 ND ND ND ND 210-138 94:06 s, v46:21_46:34_97:20 69:31 70.74 ND 0.163 ND 121-175 93:07 s, v 79:12_85:790:10 70.69 60.9 0.1785 ND (0.007) 211-212 92:08 s 88:02_122:01 98:01 ND68.62 ND 0.052 213-214 92:08 s, v 51:35_66:14_76:32 70:30 55.83 67.290.832 0.861 215-216 92:08 s 66:35 66:34 ND ND ND ND 115-217 91:09 s, v79:2_97:2 98:02 67.79 58.76 0.127 7.89 115-218 91:09 s, v 62:11_94:393:07 67.79 ND 0.127 ND 123-219 91:09 s, v 80:13_91:7 90:10 67.79 60.90.127 ND 117-217 90:10 s, v 79:2_97:2 98:02 69.62 58.76 0.101 7.89117-218 90:10 s, v 62:11_94:3 93:07 69.62 ND 0.101 ND 220-199 90:10 s, v68:15_74:21 80:20 59.74 68.63 0.55 1.78 118-143 89:11 s, v 94:1_97:199:01 70.69 58.76 0.1785 7.89 (0.007) 118-144 89:11 s, v 105:3_57:5_97:197:03 70.69 ND 0.1785 ND (0.007) 221-222 88:12 s 71:42 63:37 ND ND ND ND223-179 87:13 s, v 64:26_69:26_69:28_75:25_77:26_(—) 75:25 ND 27.52 NDNB 78:26_83:22_86:16_95:8 224-225 86:14 s 52:29 64:36 ND 68.62 ND 0.052226-227 85:15 s, v 73:19_91:7 87:13 66.94 ND 0.0134 ND 226-228 85:15 s,v 67:31_72:20 74:26 66.94 61.43 0.0134 2.51 229-230 84:16 s, v 76:2_79:198:02 69.875 61.7 NB 0.42 (0.03) 201-83  84:16 v 84:03 96:04 ND 68.37 ND0.0828 231-232 82:18 s 92:07 93:07 ND ND ND ND 233-131 80:20 s, v81:16_88:10 87:13 65.72 68.63 0.0348 1.78 234-235 79:21 s, v 102:1_106:299:01 ND 66.67 ND 0.0511 236-237 79:21 s 108:02 98:02 ND ND ND ND234-238 79:21 s 98:04 97:03 ND 70.11 ND 0.0816 239-240 79:21 s 79:1089:11 65.72 ND 0.0348 ND 241-242 79:21 s, v 36:25_41:25_58:7_67:6 78:2260.9 56.17 ND ND 239-243 79:21 s, v 54:24_69:18 75:25 65.72 62.29 0.03480.984 242-244 78:22 s, v 53:17_59:27_67:19 75:25 56.17 ND ND ND 179-24575:25 s, v 79:3_89:1 98:02 27.52 67.82 NB 3.4 (2.52) 246-225 74:26 s52:29 64:36 ND 68.62 ND 0.052 247-248 73:27 s, v 50:32_56:22 67:33 27.52ND NB ND 125-219 72:28 s, v 80:13_91:7 90:10 69.62 60.9 0.101 ND 249-25067:33 s, v 43:26_43:27_53:19 62:38 66.54 ND 0.197 ND 251-240 66:34 s79:10 89:11 68.1 ND 0.1247 ND (0.0646) 252-227 66:34 s, v 73:19_91:787:13 ND ND ND ND 251-243 66:34 s, v 54:24_69:18 75:25 68.1 62.29 0.12470.984 (0.0646) 252-228 66:34 s, v 67:31_72:20 74:26 ND 61.43 ND 2.51253-120 65:35 s, v 61:35_74:19 72:28 ND 67.29 ND 0.861 254-255 64:36 s,v 73:2_41:33 88:12 63.91 ND 0.0792 ND 250-256 62:38 s, v59:20_63:19_74:11 77:23 ND ND ND ND

TABLE 15 Unique identifier set Fab Region Design Type H1_mutationL1_mutation H2_mutation L2_mutation 257-258 constant electrostaticL143E_K145T Q124R_Q160K_T178R S186R Q124E_Q160E_T180E 259-260 constantelectrostatic L143E_K145T Q124K_T178R S186R Q124E_Q160E_T180E 261-262constant combination L124E_H172R V133G_S176R L124R_H172AV133G_S174W_S176D (electrostatic + steric) 263-264 constantelectrostatic L124E V133G_S176R L124R V133G_S176D 265-266 constantelectrostatic L143E_K145T Q124K_T178R S186R Q124R 267-268 constantcombination L124E_H172R V133G_S176R L124R_H172A V133A_S174W_S176D(electrostatic + steric) 269-270 constant combination L124E_H172WV133G_S176R L124R_H172T V133G_S174R_S176D (electrostatic + steric)271-272 constant combination L124E_H172R V133A_S176K L124R_H172AV133A_S174W_S176D (electrostatic + steric) 273-274 variable combinationQ39E Q38R_F98W Q39R_F100W_W103F Q38E_F98M (electrostatic + steric)275-276 constant combination L124E_H172W V133A_S176K L124R_H172TV133G_S174R_S176D (electrostatic + steric) 277-278 constantelectrostatic L143E_K145T Q124R L143R Q124E 277-279 constantelectrostatic L143E_K145T Q124R WT Q124E 277-280 constant electrostaticL143E_K145T Q124E S186R Q124E 277-281 constant electrostatic L143E_K145TQ124R L143K Q124E 282-283 constant electrostatic L143E_K145T Q124R S186RT178E 284-285 constant electrostatic L143E_K145T Q124R S186RQ124E_Q160E_T180E 286-287 constant combination L124E_H172W V133G_S176RL124R_H172T V133A_S174R_S176D (electrostatic + steric) 288-289 constantelectrostatic L124E V133G_S176R L124R V133A_S176D and steric 290-291constant electrostatic L143E_K145T_S188L Q124K L143K Q124E 290-292constant electrostatic L143E_K145T_S188L Q124K S186R Q124E 290-293constant electrostatic L143E_K145T_S188L Q124K L143R Q124E 294-295constant electrostatic L124E V133A_S176K L124R V133G_S176D and steric296-297 variable steric F100W F98M W103F Y36W 298-297 variable stericF100W_W103F F98M W103F Y36W 299-300 variable steric L45A P44F V37W F98A301-302 variable electrostatic Q39E Q38N_T85R Q39R Q38E_T85E 303-278constant electrostatic L143E_K145T_S188L Q124R L143R Q124E 303-279constant electrostatic L143E_K145T_S188L Q124R WT Q124E 303-280 constantelectrostatic L143E_K145T_S188L Q124R S186R Q124E 303-281 constantelectrostatic L143E_K145T_S188L Q124R L143K Q124E Screening Screeningonly/Verification only/Verification only/Screening only/ScreeningPresence of and verification Normalized and verification Unique H-Ldisulfide data for H1- medain data for H2- identifier bond (C233-L1:H1-L2 H1- L2:H2-L1 set C214) (y/n) (s/v/s_v) H1-L1:H1-L2 L1:H1-L2(s/v/s_v) H2-L2:H2-L1 257-258 n s 93:3  97:3  v 94:11 259-260 n v 90:6 94:6  v 91:29 261-262 n v_s 99:8_94:8 93:7  v_s 48:21_58:15 263-264 nv_s 95:5_68:25 88:12 v_s 70:29_101:9 265-266 n v 90:13 87:13 v 96:6 267-268 n v_s_s 86:12_80:12_85:19 87:13 v_s 52:22_51:16 269-270 n v_s82:15_76:13 85:15 v_s 89:13_84:11 271-272 n v_s 90:19_74:16 82:18 v_s49:35_46:19 273-274 n v_s 73:11_54:19 81:19 v_s 56:45_80:4 275-276 n v_s75:18_60:15 80:20 v_s 82:21_79:18 277-278 n v 93:26 78:22 s 77:20277-279 n v 93:26 78:22 v_v_s 81:24_76:22_77:8 277-280 n v 93:26 78:22 v86:30 277-281 n v 93:26 78:22 v_s_s 70:29_97:10_31:35 282-283 n v 92:2877:23 v 68:30 284-285 n v 82:26 76:24 v 94:9  286-287 n v_s 68:30_64:2172:28 v_s 83:18_83:14 288-289 n v_s 58:46_59:16 68:32 v_s 83:12_81:8290-291 n v_s 75:26_47:32 68:32 s 88:6  290-292 n v_s 75:26_47:32 68:32v 87:7  290-293 n v_s 75:26_47:32 68:32 v_s 84:20_87:25 294-295 n v_s69:35_49:30 64:36 v_s 68:40_42:22 296-297 n v_s 70:38_65:38 64:36 v_s53:41_77:43 298-297 n v_s 67:45_62:36 62:38 v_s 53:41_77:43 299-300 nv_s 59:48_60:33 60:40 v_s 92:11_81:17 301-302 n v_s_s 66:44_45:31_62:3160:40 v_s 64:39_86:27 303-278 n v_s_s 70:52_59:41_76:27 59:41 s 77:20303-279 n v_s_s 70:52_59:41_76:27 59:41 v_v_s 81:24_76:22_77:8 303-280 nv_s_s 70:52_59:41_76:27 59:41 v 86:30 303-281 n v_s_s 70:52_59:41_76:2759:41 v_s_s 70:29_97:10_31:35 Unique Normalized identifier median H2-H1-L1 Tm H2-L2 Tm set L2:H2-L1 (° C.) (° C.) 257-258 89:11 ND ND 259-26076:24 ND ND 261-262 75:25 77.7  76   263-264 84:16 76.96 ND 265-26694:6  ND 267-268 73:27 77.7  ND 269-270 88:12 76.43 77.04 271-272 65:3576.49 ND 273-274 84:16 74.32 70.41 275-276 80:20 75.62 77.04 277-27879:21 ND 76.56 277-279 78:22 ND ND 277-280 74:26 ND ND 277-281 71:29 NDND 282-283 70:30 ND 76.03 284-285 92:8  ND ND 286-287 84:16 76.43 77.12288-289 89:11 76.96 77.45 290-291 93:7  76.95 ND 290-292 92:8  76.95 ND290-293 79:21 76.95 76.56 294-295 64:36 76.07 ND 296-297 61:39 72.2773.15 298-297 61:39 71.38 73.15 299-300 86:14 71.4   78.545 301-30269:31 ND 71.37 303-278 79:21 ND 76.56 303-279 78:22 ND ND 303-280 74:26ND ND 303-281 71:29 ND ND

TABLE 16 Unique REF_WT Design H1_(—) identifier or VAR Type AbH1_mutation L1_Ab L1_mutation A TRAS TRAS  58 electrostatic TRASL143E_K145T TRAS Q124R 304* combination TRAS A139W_S186K TRASF116A_Q124E_L135A_T180E (steric + electrostatic) 305* combination TRASV37W TRAS F98A (steric + electrostatic)  26 combination TRAS V37W_Q39ETRAS Q38R_F98A (steric + electrostatic)  57 electrostatic TRASL143K_D146G TRAS Q124E_V133D 306* combination TRASA139G_K145T_D146G_Q179E_V190A TRAS L135W (steric + electrostatic) 307*combination TRAS Q39R TRAS Q38E (steric + electrostatic)  93 combinationTRAS Q39R TRAS Q38E_F98W (steric + electrostatic)  72 electrostatic TRASD146G_Q179R TRAS Q124E_Q160E_T178D  3 steric TRAS F174V_P175S_S188G TRASS176L 108 steric TRAS A139W TRAS F116A_L135A 308* steric TRAS L124W TRASF118A  52 electrostatic TRAS Q39E TRAS Q38R 309* electrostatic TRASV37E_M100D TRAS Q89R_F98W  54 electrostatic TRAS WT TRAS WT  65electrostatic TRAS K145T_Q179D_S188L TRAS Q160K_T178R  19 steric TRASS188L_V190Y TRAS V133S 107 steric TRAS A139G_V190A TRAS L135W  51electrostatic TRAS Q39R TRAS Q38E 310* electrostatic TRAS WT TRAS WT311* electrostatic TRAS WT TRAS WT  53 electrostatic TRAS Q39R TRAS Q38E312* steric TRAS WT TRAS WT B TRAS TRAS C TRAS TRAS  67 electrostaticTRAS K145T_Q179D_S188F TRAS V133A_Q160K_T178R  58 electrostatic TRASL143E_K145T TRAS Q124R  66 electrostatic TRAS L143A_D146G_Q179R TRASQ124E_V133W_Q160E_T180E 304* combination TRAS A139W_S186K TRASF116A_Q124E_L135A_T180E (steric + electrostatic) 165 combination TRASA139W TRAS F116A_L135A (steric + electrostatic) 307* combination TRASQ39R TRAS Q38E (steric + electrostatic)  26 combination TRAS V37W_Q39ETRAS Q38R_F98A (steric + electrostatic) 313* steric TRAS V37W TRAS F98A306* combination TRAS A139G_K145T_D146G_Q179E_V190A TRAS L135W (steric +electrostatic)  57 electrostatic TRAS L143K_D146G TRAS Q124E_V133D  93combination TRAS Q39R TRAS Q38E_F98W (steric + electrostatic)  3 stericTRAS F174V_P175S_S188G TRAS S176L 108 steric TRAS A139W TRAS F116A_L135A314* combination TRAS A139V_K145L_Q179E_S188G_V190S TRASF116A_S131K_V133G_S176F_T178A (steric + electrostatic) 164 combinationTRAS A139G_K145L_Q179E_V190A TRAS S131R_L135W (steric + electrostatic) 72 electrostatic TRAS D146G_Q179R TRAS Q124E_Q160E_T178D 315*combination TRAS A139W_S186K_S188A TRAS F118W_V133S_S176A_T180E(steric + electrostatic)  51 electrostatic TRAS Q39R TRAS Q38E 309*electrostatic TRAS V37E_M100D TRAS Q89R_F98W 316* electrostatic TRASV37E TRAS Q89R_F98T 317* electrostatic TRAS V37S_S93K TRAS F98Y 318*electrostatic TRAS V37E_M100D TRAS Q89R_F98W  19 steric TRAS S188L_V190YTRAS V133S 107 steric TRAS A139G_V190A TRAS L135W 102 electrostatic TRASWT TRAS WT D PERT TRAS  58 electrostatic PERT L143E_K145T PERT Q124R304* combination PERT A139W_S186K PERT F116A_(—) (steric +Q124E_L135A_T180E electrostatic)  26 combination PERT V37W_Q39E PERTQ38R_F98A (steric + electrostatic)  72 electrostatic PERT D146G_Q179RPERT Q124E_Q160E_T178D  3 steric PERT F174V_P175S_S188G PERT S176L 108steric PERT A139W PERT F116A_L135A 308* steric PERT L124W PERT F118A  52electrostatic PERT Q39E PERT Q38R 319* electrostatic PERT V37E_F100DPERT Q89R_F98W E PERT PERT  72 electrostatic PERT D146G_Q179R PERTQ124E_Q160E_T178D  58 electrostatic PERT L143E_K145T PERT Q124R  3steric PERT F174V_P175S_S188G PERT S176L 304* combination PERTA139W_S186K PERT F116A_Q124E_L135A_T180E (steric + electrostatic) 308*steric PERT L124W PERT F118A  52 electrostatic PERT Q39E PERT Q38R 319*electrostatic PERT V37E_F100D PERT Q89R_F98W 305* combination PERT V37WPERT F98A (steric + electrostatic)  26 combination PERT V37W_Q39E PERTQ38R_F98A (steric + electrostatic)  65 electrostatic PERTK145T_Q179D_S188L PERT Q160K_T178R  57 electrostatic PERT L143K_D146GPERT Q124E_V133D 107 steric PERT A139G_V190A PERT L135W 306* combinationPERT A139G_K145T_D146G_Q179E_V190A PERT L135W (steric + electrostatic) 51 electrostatic PERT Q39R PERT Q38E 310* electrostatic PERT WT PERT WT311* electrostatic PERT WT PERT WT 307* combination PERT Q39R PERT Q38E(steric + electrostatic)  53 electrostatic PERT Q39R PERT Q38E 312*steric PERT WT PERT WT  93 combination PERT Q39R PERT Q38E_F98W(steric + electrostatic) 108 steric PERT A139W PERT F116A_L135A  19steric PERT S188L_V190Y PERT V133S 320* steric PERT L124S PERT WT F PERTPERT G PERT PERT  58 electrostatic PERT L143E_K145T PERT Q124R  3 stericPERT F174V_P175S_S188G PERT S176L 108 steric PERT A139W PERT F116A_L135A 66 electrostatic PERT L143A_D146G_Q179R PERT Q124E_V133W_Q160E_T180E304* combination PERT A139W_S186K PERT F116A_Q124E_L135A_T180E (steric +electrostatic) 315* combination PERT A139W_S186K_S188A PERTF118W_V133S_S176A_T180E (steric + electrostatic) 165 combination PERTA139W PERT F116A_L135A (steric + electrostatic)  51 electrostatic PERTQ39R PERT Q38E 307* combination PERT Q39R PERT Q38E (steric +electrostatic)  26 combination PERT V37W_Q39E PERT Q38R_F98A (steric +electrostatic)  65 electrostatic PERT K145T_Q179D_S188L PERT Q160K_T178R306* combination PERT A139G_K145T_D146G_Q179E_V190A PERT L135W (steric +electrostatic)  57 electrostatic PERT L143K_D146G PERT Q124E_V133D  93combination PERT Q39R PERT Q38E_F98W (steric + electrostatic) 314*combination PERT A139V_K145L_Q179E_S188G_V190S PERTF116A_S131K_V133G_S176F_T178A (steric + electrostatic)  72 electrostaticPERT D146G_Q179R PERT Q124E_Q160E_T178D  53 electrostatic PERT Q39R PERTQ38E 321* steric PERT V37W_L45W PERT Y87A_F98A  52 electrostatic PERTQ39E PERT Q38R  48 electrostatic PERT WT PERT WT 322* steric PERT L45APERT Y87W_G101I 323* steric PERT W103V PERT P44W_Q89W_F98A  19 stericPERT S188L_V190Y PERT V133S 107 steric PERT A139G_V190A PERT L135W 320*steric PERT L124S PERT WT 102 electrostatic PERT WT PERT WT H D3H44D3H44 I D3H44 D3H44  57 electrostatic D3H44 L143K_D146G D3H44Q124E_V133D  72 electrostatic D3H44 D146G_Q179R D3H44 Q124E_Q160E_T178D 66 electrostatic D3H44 L143A_D146G_Q179R D3H44 Q124E_V133W_Q160E_T180E182 electrostatic D3H44 V37E_F100D D3H44 L89R_F98W  47 electrostaticD3H44 V37E_F100D D3H44 L89R_F98W 324* steric D3H44 V37W_F100W D3H44 F98AJ D3H44 D3H44 K D3H44 D3H44  26 combination D3H44 V37W_Q39E D3H44Q38R_F98A (steric + electrostatic)  66 electrostatic D3H44L143A_D146G_Q179R D3H44 Q124E_V133W_Q160E_T180E  57 electrostatic D3H44L143K_D146G D3H44 Q124E_V133D 165 combination D3H44 A139W D3H44F116A_L135A (steric + electrostatic) 313* steric D3H44 V37W D3H44 F98A305* combination D3H44 V37W D3H44 F98A (steric + electrostatic)  93combination D3H44 Q39R D3H44 Q38E_F98W (steric + electrostatic) 307*combination D3H44 Q39R D3H44 Q38E (steric + electrostatic) 310*electrostatic D3H44 WT D3H44 WT 311* electrostatic D3H44 WT D3H44 WT 182electrostatic D3H44 V37E_F100D D3H44 L89R_F98W 325* steric D3H44 V37WD3H44 F98A  47 electrostatic D3H44 V37E_F100D D3H44 L89R_F98W 324*steric D3H44 V37W_F100W D3H44 F98A Unique identifier L1_tag L2_AbL2_mutation L2_tag FLAG TRAS HA  58 FLAG TRAS Q124E_V133D HA 304* FLAGTRAS L135W HA 305* FLAG TRAS Q38E HA  26 FLAG TRAS Q38E_F98W HA  57 HATRAS Q124R FLAG 306* HA TRAS F116A_Q124E_L135A_T180E FLAG 307* HA TRASF98A FLAG  93 HA TRAS Q38R_F98A FLAG  72 FLAG TRAS Q160K_T178R HA  3FLAG TRAS V133S HA 108 FLAG TRAS L135W HA 308* FLAG TRAS WT HA  52 FLAGTRAS Q38E HA 309* FLAG TRAS WT HA  54 FLAG TRAS Q38E HA  65 HA TRASQ124E_Q160E_T178D FLAG  19 HA TRAS S176L FLAG 107 HA TRAS F116A_L135AFLAG  51 HA TRAS Q38R FLAG 310* HA TRAS Q89R_F98W FLAG 311* HA TRASQ89R_F98T FLAG  53 HA TRAS WT FLAG 312* HA TRAS F98A FLAG FLAG D3H44 HAHA D3H44 FLAG  67 FLAG D3H44 Q124E_V133W_Q160E_T180E HA  58 FLAG D3H44Q124E_V133D HA  66 FLAG D3H44 V133A_Q160K_T178R HA 304* FLAG D3H44 L135WHA 165 FLAG D3H44 S131R_L135W HA 307* FLAG D3H44 F98A HA  26 FLAG D3H44Q38E_F98W HA 313* FLAG D3H44 P44W HA 306* HA D3H44F116A_Q124E_L135A_T180E FLAG  57 HA D3H44 Q124R FLAG  93 HA D3H44Q38R_F98A FLAG  3 FLAG D3H44 V133S HA 108 FLAG D3H44 L135W HA 314* FLAGD3H44 F118W_V133S_S176A_T180E HA 164 FLAG D3H44 F116A_L135A HA  72 FLAGD3H44 Q160K_T178R HA 315* FLAG D3H44 F116A_S131K_V133G_S176F_T178A HA 51 FLAG D3H44 Q38R HA 309* FLAG D3H44 WT HA 316* FLAG D3H44 WT HA 317*FLAG D3H44 L89R_F98W HA 318* FLAG D3H44 F98Y HA  19 HA D3H44 S176L FLAG107 HA D3H44 F116A_L135A FLAG 102 HA D3H44 L89R_F98T FLAG FLAG TRAS HA 58 FLAG TRAS Q124E_V133D HA 304* FLAG TRAS L135W HA  26 FLAG TRASQ38E_F98W HA  72 FLAG TRAS Q160K_T178R HA  3 FLAG TRAS V133S HA 108 FLAGTRAS L135W HA 308* FLAG TRAS WT HA  52 FLAG TRAS Q38E HA 319* FLAG TRASWT HA FLAG PERT HA  72 FLAG PERT Q160K_T178R HA  58 FLAG PERTQ124E_V133D HA  3 FLAG PERT V133S HA 304* FLAG PERT L135W HA 308* FLAGPERT WT HA  52 FLAG PERT Q38E HA 319* FLAG PERT WT HA 305* FLAG PERTQ38E HA  26 FLAG PERT Q38E_F98W HA  65 HA PERT Q124E_Q160E_T178D FLAG 57 HA PERT Q124R FLAG 107 HA PERT F116A_L135A FLAG 306* HA PERTF116A_Q124E_L135A_T180E FLAG  51 HA PERT Q38R FLAG 310* HA PERTQ89R_F98W FLAG 311* HA PERT Q89R_F98T FLAG 307* HA PERT F98A FLAG  53 HAPERT WT FLAG 312* HA PERT F98A FLAG  93 HA PERT Q38R_F98A FLAG 108 FLAGPERT L135W HA  19 HA PERT S176L FLAG 320* HA PERT F118A FLAG FLAG D3H44HA HA D3H44 FLAG  58 FLAG D3H44 Q124E_V133D HA  3 FLAG D3H44 V133S HA108 FLAG D3H44 L135W HA  66 FLAG D3H44 V133A_Q160K_T178R HA 304* FLAGD3H44 L135W HA 315* FLAG D3H44 F116A_S131K_V133G_S176F_T178A HA 165 FLAGD3H44 S131R_L135W HA  51 FLAG D3H44 Q38R HA 307* FLAG D3H44 F98A HA  26FLAG D3H44 Q38E_F98W HA  65 HA D3H44 Q124E_Q160E_T178D FLAG 306* HAD3H44 F116A_Q124E_L135A_T180E FLAG  57 HA D3H44 Q124R FLAG  93 HA D3H44Q38R_F98A FLAG 314* FLAG D3H44 F118W_V133S_S176A_T180E HA  72 FLAG D3H44Q160K_T178R HA  53 FLAG D3H44 WT HA 321* FLAG D3H44 Y87W_G101I HA  52FLAG D3H44 Q38E HA  48 FLAG D3H44 L89R_F98W HA 322* FLAG D3H44 Y87A_F98AHA 323* FLAG D3H44 F98A HA  19 HA D3H44 S176L FLAG 107 HA D3H44F116A_L135A FLAG 320* HA D3H44 F118A FLAG 102 HA D3H44 L89R_F98T FLAGFLAG TRAS HA HA TRAS FLAG  57 HA TRAS Q124R FLAG  72 FLAG TRASQ160K_T178R HA  66 HA TRAS V133A_Q160K_T178R FLAG 182 HA TRAS F98Y FLAG 47 HA TRAS WT FLAG 324* HA TRAS P44W_Q89W_F98A FLAG FLAG PERT HA HAPERT FLAG  26 FLAG PERT Q38E_F98W HA  66 HA PERT V133A_Q160K_T178R FLAG 57 HA PERT Q124R FLAG 165 HA PERT S131R_L135W FLAG 313* HA PERT P44WFLAG 305* HA PERT Q38E FLAG  93 HA PERT Q38R_F98A FLAG 307* HA PERT F98AFLAG 310* HA PERT Q89R_F98W FLAG 311* HA PERT Q89R_F98T FLAG 182 HA PERTF98Y FLAG 325* HA PERT F98W FLAG  47 HA PERT WT FLAG 324* HA PERTP44W_Q89W_F98A FLAG Presence of H-L disulfide bond (C233- ObservedNumber of Normalized Unique C214) Trends for screening Median identifier(y/n) REF_WT experiments H1-L1:H1-L2 H1-L1:H1-L2 n Apparent tag 666:34_57:36_66:43_54:36_58:43_58:43 61:39 dependence. Likely similarissue with HA-tag as with PERT system, but to a lesser extent  58 n 2104:1_77:2 99:01 304* n 2 103:1_92:1 99:01 305* n 2 73:17_73:20 80:20 26 n 2 87:1_79:1 99:01  57 n 2 89:1_89:1 99:01 306* n 2 76:1_69:2 98:02307* n 2 92:1_74:2 98:02  93 n 2 103:1_73:1 99:01  72 n 1 66:13 84:16  3n 1 92:02 98:02 108 n 1 70:14 83:17 308* n 1 66:28 70:30  52 n 1 73:1880:20 309* n 1 78:17 82:18  54 n 1 63:15 81:19  65 n 1 56:33 63:37  19 n1 59:35 63:37 107 n 1 58:19 75:25  51 n 1 55:35 61:39 310* n 1 87:0694:06 311* n 1 92:01 99:01  53 n 1 59:32 65:35 312* n 1 84:05 94:06 nPreference of 5 70:16_84:21_84:21_72:44_66:50 80:20 n H_TRAS for 5107:15_107:18_86:17_89:18_61:19 84:16 L_TRAS over L_D3H44  67 n 2115:1_104:1 99:01  58 n 2 105:1_94:1 99:01  66 n 2 102:1_83:2 99:01 304*n 2 98:1_68:1 99:01 165 n 2 103:1_83:1 99:01 307* n 2 102:2_73:9 96:04 26 n 2 91:2_87:11 95:05 313* n 2 92:3_91:11 94:06 306* n 2 108:1_90:199:01  57 n 2 104:1_88:1 99:01  93 n 2 96:1_87:1 99:01  3 n 1 77:0297:03 108 n 1 80:01 99:01 314* n 1 88:01 99:01 164 n 1 84:01 99:01  72 n1 72:01 99:01 315* n 1 107:03  97:03  51 n 1 73:12 86:14 309* n 1110:05  95:05 316* n 1 97:10 91:09 317* n 1 72:12 86:14 318* n 1 99:0794:06  19 n 1 92:13 87:13 107 n 1 86:02 98:02 102 n 1 111:01  99:01 nLikely no 5 68:43_66:42_55:56_56:58_48:60 49:51 preference of H_PERT forL_PERT over L_TRAS  58 n 2 84:5_70:23 88:12 304* n 2 77:22_75:23 77:23 26 n 2 82:10_79:12 88:12  72 n 1 83:13 87:13  3 n 1 72:12 86:14 108 n 178:32 71:29 308* n 1 62:36 64:36  52 n 1 62:35 64:36 319* n 1 92.0199:01 n Apparent tag 1081:20_79:20_70:29_78:34_78:47_57:35_56:36_65:53_65:53_53:50 62:38dependence observed. Observed ratios are likely due to HA-tag cleavagerather than tag interference with pairing  72 n 2 101:1_79:8 97:03  58 n2 99:1_97:1 99:01  3 n 2 102:1_94:1 99:01 304* n 2 97:1_101:3 98:02 308*n 2 72:21_69:23 76:24  52 n 2 82:8_96:10 91:09 319* n 2 76:1_94:2 98:02305* n 2 90:9_79:17 87:13  26 n 2 101:1_98:1 99:01  65 n 2 97:8_73:1389:11  57 n 2 110:1_94:1 99:01 107 n 2 92:3_78:2 97:03 306* n 2102:1_96:1 99:01  51 n 2 125:6_75:15 91:09 310* n 2 110:9_75:13 89:11311* n 2 126:2_88:3 98:02 307* n 2 124:1_95:1 99:01  53 n 2 112:8_70:3385:15 312* n 2 105:20_66:35 76:24  93 n 2 103:1_82:1 99:01 108 n 1 63:2770:30  19 n 2 81:45_60:41 62:38 320* n 2 53:34_63:44 60:40 n Preferenceof 7 106:1_104:1_123:2_105:6_126:9_102:7_113:15 94:06 n H_PERT for 598:26_68:23_68:25_65:44_67:46 73:27 L_PERT over L_D3H44  58 n 2112:1_99:10 97:03  3 n 6 111:1_105:1_104:1_97:1_94:1_90:1 99:01 108 n 696:1_92:1_103:1_109:2_113:3_102:3 99:01  66 n 2 117:1_110:1 99:01 304* n2 106:1_84:1 99:01 315* n 2 111:1_115:25 96:04 165 n 2 109:1_109:1297:03  51 n 6 106:1_106:2_98:2_97:2_110:3_97:3 98:02 307* n 6113:1_97:1_83:1_108:2_100:2_115:2 99:01  26 n 2 101:1_84:1 99:01  65 n 298:1_63:1 99:01 306* n 2 119:1_112:1 99:01  57 n 2 125:1_107:1 99:01  93n 2 89:1_88:6 97:03 314* n 1 108:01  99:01  72 n 1 95:01 99:01  53 n 1116:02  98:02 321* n 1 90:03 97:03  52 n 1 105:01  99:01  48 n 1 112:01 99:01 322* n 1 97:01 99:01 323* n 1 71:01 99:01  19 n 1 101:15  87:13107 n 1 102:01  99:01 320* n 1 79:06 93:07 102 n 1 113:03  97:03 nPreference of 5 36:59_36:59_35:86_31:87_20:67 29:71 n H_D3H44 for 550:58_49:61_34:68_21:71_21:76 33:67 L_TRAS over L_D3H44  57 n 2100:1_85:4 98:02  72 n 1 67:36 65:35  66 n 1 92:20 82:18 182 n 1 83:0397:03  47 n 1 84:02 97:03 324* n 1 77:25 75:25 n Inconsistency 592:4_88:8_58:52_43:60_47:70 53:47 n in observed 734:65_29:70_27:67_29:73_27:73_2:101_1:98 29:71 ratios of H_D3H44 towardsFLAG_L_D3H44 or HA_L_PERT likely due to HA-tag issue, as preference ofH_D3H44 for FLAG_L_PERT over HA_L_D3H44 is consistent  26 n 2 96:1_69:3294:06  66 n 2 82:4_92:24 90:10  57 n 2 90:3_85:4 96:04 165 n 297:4_58:30 87:13 313* n 2 108:1_82:27 94:06 305* n 6102:3_99:3_101:4_121:5_110:10_85:24 96:04  93 n 2 76:24_70:31 73:27 307*n 2 80:34_70:33 69:31 310* n 1 74:39 65:35 311* n 1 75:25 75:25 182 n 174:03 96:04 325* n 6 74:24_84:29_74:26_78:32 73:27 78:36_66:51  47 n 182:02 97:03 324* n 1 92:17 84:16 Normalized REF_WT Δ(VAR- H1-L1:H1-L2Median H1-L1:H1-L2 (REF_WT) Unique L2 Scalar REF_WT REF_WT ScalarH1-L1:H1-L2 identifier (Median) for VAR H1-L1:H1-L2 (Median) Scalar0.4295 N/A N/A N/A N/A  58 4.288 A 61:39 0.4295 3.86 304* 4.548 A 61:390.4295 4.12 305* 1.402 A 61:39 0.4295 0.97  26 4.423 A 61:39 0.4295 3.99 57 4.488 A 39:61 −0.4295 4.92 306* 3.797 A 39:61 −0.4295 4.23 307*4.1395 A 39:61 −0.4295 4.57  93 4.4615 A 39:61 −0.4295 4.89  72 1.644 A61:39 0.4295 1.21  3 4.016 A 61:39 0.4295 3.59 108 1.584 A 61:39 0.42951.15 308* 0.864 A 61:39 0.4295 0.43  52 1.413 A 61:39 0.4295 0.98 309*1.499 A 61.39 0.4295 1.07  54 1.459 A 61:39 0.4295 1.03  65 0.529 A39:61 −0.4295 0.96  19 0.527 A 39:61 −0.4295 0.96 107 1.122 A 39:61−0.4295 1.55  51 0.451 A 39:61 −0.4295 0.88 310* 2.7 A 39:61 −0.42953.13 311* 4.52 A 39:61 −0.4295 4.95  53 0.613 A 39:61 −0.4295 1.04 312*2.814 A 39:61 −0.4295 3.24 1.369 N/A N/A N/A N/A 1.622 N/A N/A N/A N/A 67 4.6925 B 80:20 1.369 3.32  58 4.5975 B 80:20 1.369 3.23  66 4.246 B80:20 1.369 2.88 304* 4.3965 B 80:20 1.369 3.03 165 4.5225 B 80:20 1.3693.15 307* 3.0565 B 80:20 1.369 1.69  26 2.9825 B 80:20 1.369 1.61 313*2.801 B 80:20 1.369 1.43 306* 4.59 C 84:16 1.622 2.97  57 4.56 C 84:161.622 2.94  93 4.5175 C 84:16 1.622 2.9   3 3.499 B 80:20 1.369 2.13 1084.377 B 80:20 1.369 3.01 314* 4.477 B 80:20 1.369 3.11 164 4.426 B 80:201.369 3.06  72 4.275 B 80:20 1.369 2.91 315* 3.426 B 80:20 1.369 2.06 51 1.825 B 80:20 1.369 0.46 309* 3.009 B 80:20 1.369 1.64 316* 2.278 B80:20 1.369 0.91 317* 1.791 B 80:20 1.369 0.42 318* 2.689 B 80:20 1.3691.32  19 1.943 C 84:16 1.622 0.32 107 4.053 C 84:16 1.622 2.43 102 4.708C 84:16 1.622 3.09 −0.026 N/A N/A N/A N/A  58 1.981 D 49:51 −0.026 2.01304* 1.221 D 49:51 −0.026 1.25  26 1.9955 D 49:51 −0.026 2.02  72 1.891D 49:51 −0.026 1.92  3 1.794 D 49:51 −0.026 1.82 108 0.897 D 49:51−0.026 0.92 308* 0.557 D 49:51 −0.026 0.58  52 0.573 D 49:51 −0.026 0.6 319* 4.524 D 49:51 −0.026 4.55 0.4975 N/A N/A N/A N/A  72 3.477 E 62:380.4975 2.98  58 4.4065 E 62:38 0.4975 3.91  3 4.585 E 62.38 0.4975 4.09304* 3.981 E 62:38 0.4975 3.48 308* 1.17 E 62:38 0.4975 0.67  52 2.323 E62:38 0.4975 1.83 319* 4.0915 E 62:38 0.4975 3.59 305* 1.9365 E 62:380.4975 1.44  26 4.6015 E 62:38 0.4975 4.1   65 2.106 E 38:62 −0.49752.6   57 4.6195 E 38:62 −0.4975 5.12 107 3.546 E 38:62 −0.4975 4.04 306*4.5905 E 38:62 −0.4975 5.09  51 2.312 E 38:62 −0.4975 2.81 310* 2.1055 E38:62 −0.4975 2.6  311* 3.7895 E 38:62 −0.4975 4.29 307* 4.688 E 38:62−0.4975 5.19  53 1.7115 E 38:62 −0.4975 2.21 312* 1.146 E 38:62 −0.49751.64  93 4.522 E 38:62 −0.4975 5.02 108 0.857 E 62:38 0.4975 0.36  190.472 E 38:62 −0.4975 0.97 320* 0.405 E 38:62 −0.4975 0.9  2.804 N/A N/AN/A N/A 0.998 N/A N/A N/A N/A  58 3.515 F 94:06 2.804 0.71  3 4.6135 F94:06 2.804 1.81 108 4.2365 F 94:06 2.804 1.43  66 4.731 F 94:06 2.8041.93 304* 4.55 F 94:06 2.804 1.75 315* 3.12 F 94:06 2.804 0.32 165 3.46F 94:06 2.804 0.66  51 3.7925 F 94:06 2.804 0.99 307* 4.2825 F 94:062.804 1.48  26 4.4435 F 94:06 2.804 1.64  65 4.3665 G 73:27 0.998 3.37306* 4.7495 G 73:27 0.998 3.75  57 4.7505 G 73:27 0.998 3.75  93 3.5745G 73:27 0.998 2.58 314* 4.684 F 94:06 2.804 1.88  72 4.328 F 94:06 2.8041.52  53 3.927 F 94:06 2.804 1.12 321* 3.496 F 94:06 2.804 0.69  524.654 F 94:06 2.804 1.85  48 4.336 F 94:06 2.804 1.53 322* 4.575 F 94:062.804 1.77 323* 4.267 F 94:06 2.804 1.46  19 1.927 G 73:27 0.998 0.93107 4.623 G 73:27 0.998 3.63 320* 2.573 G 73:27 0.998 1.58 102 3.52 G73:27 0.998 2.52 −0.894 N/A N/A N/A N/A −0.701 N/A N/A N/A N/A  57 3.832I 33:67 −0.701 4.53  72 0.633 H 29:71 −0.894 1.53  66 1.549 I 33:67−0.701 2.25 182 3.41 I 33:67 −0.701 4.11  47 3.55 I 33:67 −0.701 4.25324* 1.116 I 33:67 −0.701 1.82 0.119 N/A N/A N/A N/A −0.909 N/A N/A N/AN/A  26 2.673 J 53:47 0.119 2.55  66 2.164 K 29:71 −0.909 3.07  573.1675 K 29:71 −0.909 4.08 165 1.8885 K 29:71 −0.909 2.8  313* 2.7655 K29:71 −0.909 3.67 305* 3.2045 K 29:71 −0.909 4.11  93 0.9895 K 29:71−0.909 1.9  307* 0.799 K 29:71 −0.909 1.71 310* 0.638 K 29:71 −0.9091.55 311* 1.109 K 29:71 −0.909 2.02 182 3.093 K 29:71 −0.909 4   325*0.979 K 29:71 −0.909 1.89  47 3.519 K 29:71 −0.909 4.43 324* 1.675 K29:71 −0.909 2.58

TABLE 17 Unique identifier REF_WT REF_WT set or VAR1 or VAR2 Design TypeH1_Ab H1_mutation L1_Ab 58-57 electrostatic PERT L143E_K145T PERT  3-19steric PERT F174V_P175S_S188G PERT 108-107 steric PERT A139W PERT308*-320* steric PERT L124W PERT 319*-310* electrostatic PERT V37E_F100DPERT 26-93 combination (steric + PERT V37W_Q39E PERT electrostatic)65-72 electrostatic PERT K145T_Q179D_S188L PERT 306*-304* combination(steric + PERT A139G_K145T_D146G_(—) PERT electrostatic) Q179E_V190A51-52 electrostatic PERT Q39R PERT 307*-305* combination (steric + PERTQ39R PERT electrostatic) 58-57 electrostatic TRAS L143E_K145T TRAS  3-19steric TRAS F174V_P175S_S188G TRAS 108-107 steric TRAS A139W TRAS309*-310* electrostatic TRAS V37E_M100D TRAS 26-93 combination (steric +TRAS V37W_Q39E TRAS electrostatic) 65-72 electrostatic TRASK145T_Q179D_S188L TRAS 306*-304* combination (steric + TRASA139G_K145T_D146G_(—) TRAS electrostatic) Q179E_V190A 51-52electrostatic TRAS Q39R TRAS 307*-305* combination (steric + TRAS Q39RTRAS electrostatic) 53-54 electrostatic TRAS Q39R TRAS I B N/A D3H44D3H44 66-67 electrostatic D3H44 L143A_D146G_Q179R D3H44 57-58electrostatic D3H44 L143K_D146G D3H44  182-317* electrostatic D3H44V37E_F100D D3H44 Presence of Unique H-L disulfide identifier bond (C233-set L1_mutation L1_tag H2_Ab H2_mutation L2_Ab L2_mutation L2_tag C214)(y/n) 58-57 Q124R FLAG PERT L143K_D146G PERT Q124E_V133D HA n  3-19S176L FLAG PERT S188L_V190Y PERT V133S HA n 108-107 F116A_L135A FLAGPERT A139G_V190A PERT L135W HA n 308*-320* F118A FLAG PERT L124S PERT WTHA n 319*-310* Q89R_F98W FLAG PERT WT PERT WT HA n 26-93 Q38R_F98A FLAGPERT Q39R PERT Q38E_F98W HA n 65-72 Q160K_T178R HA PERT D146G_Q179R PERTQ124E_Q160E_T178D FLAG n 306*-304* L135W HA PERT A139W_S186K PERTF116A_Q124E_L135A_(—) FLAG n T180E 51-52 Q38E HA PERT Q39E PERT Q38RFLAG n 307*-305* Q38E HA PERT V37W PERT F98A FLAG n 58-57 Q124R FLAGTRAS L143K_D146G TRAS Q124E_V133D HA n  3-19 S176L FLAG TRAS S188L_V190YTRAS V133S HA n 108-107 F116A_L135A FLAG TRAS A139G_V190A TRAS L135W HAn 309*-310* Q89R_F98W FLAG TRAS WT TRAS WT HA n 26-93 Q38R_F98A FLAGTRAS Q39R TRAS Q38E_F98W HA n 65-72 Q160K_T178R HA TRAS D146G_Q179R TRASQ124E_Q160E_T178D FLAG n 306*-304* L135W HA TRAS A139W_S186K TRASF116A_Q124E_L135A_(—) FLAG n T180E 51-52 Q38E HA TRAS Q39E TRAS Q38RFLAG n 307*-305* Q38E HA TRAS V37W TRAS F98A FLAG n 53-54 Q38E HA TRASWT TRAS WT FLAG n HA TRAS TRAS FLAG n 66-67 Q124E_V133W_Q160E_(—) HATRAS K145T_Q179D_S188F TRAS V133A_Q160K_T178R FLAG n T180E 57-58Q124E_V133D HA TRAS L143E_K145T TRAS Q124R FLAG n  182-317* L89R_F98W HATRAS V37S_S93K TRAS F98Y FLAG n Normalized REF_WT for Median VAR1 H1- Δ(VAR1- Unique Normalized H1-L1:H1- REF_WT REF_WT for L1:H1-L2 REF_WT)H1- identifier Observed Trends Median H1- L2 Scalar for VAR1 H1- ScalarL1:H1-L2 set for REF_WTs H1-L1:H1-L2 L1:H1-L2 (Median) VAR1 L1:H1-L2(Median) Scalar 58-57 99:1_97:1 99:01 4.4065 E 62:38 0.4975 3.91  3-19102:1_94:1 99:01 4.585 E 62:38 0.4975 4.09 108-107 63:27 70:30 0.857 E62:38 0.4975 0.36 308*-320* 72:21_69:23 76:24 1.17 E 62:38 0.4975 0.67319*-310* 76:1_94:2 98:02 4.0915 E 62:38 0.4975 3.59 26-93 101:1_98:199:01 4.6015 E 62:38 0.4975 4.1 65-72 97:8_73:13 89:11 2.106 E 38:62−0.4975 2.6 306*-304* 102:1_96:1 99:01 4.5905 E 38:62 −0.4975 5.09 51-52125:6_75:15 91:09 2.312 E 38:62 −0.4975 2.81 307*-305* 124:1_95:1 99:014.688 E 38:62 −0.4975 5.19 58-57 104:1_77:2 99:01 4.288 A 61:39 0.42953.86  3-19 92:02 98:02 4.016 A 61:39 0.4295 3.59 108-107 70:14 83:171.584 A 61:39 0.4295 1.15 309*-310* 78:17 82:18 1.499 A 61:39 0.42951.07 26-93 87:1_79:1 99:01 4.423 A 61:39 0.4295 3.99 65-72 56:33 63:370.529 A 39:61 −0.4295 0.96 306*-304* 76:1_69:2 98:02 3.797 A 39:61−0.4295 4.23 51-52 55:35 61:39 0.451 A 39:61 −0.4295 0.88 307*-305*92:1_74:2 98:02 4.1395 A 39:61 −0.4295 4.57 53-54 59:32 65:35 0.613 A39:61 −0.4295 1.04 Preference of 50:58_49:61_(—) 33:67 −0.701 N/A N/AN/A N/A H_D3H44 for L_TRAS 34:68_21:71_21:76 over L_D3H44 and H_TRAS forL_TRAS over L_D3H44 66-67 92:20 82:18 1.549 I 33:67 −0.701 2.25 57-58100:1_85:4 98:02 3.832 I 33:67 −0.701 4.53  182-317* 83:03 97:03 3.41 I33:67 −0.701 4.11 Normalized REF_WT for Median VAR2 H2- Δ (VAR2- UniqueNormalized H2-L2:H2- REF_WT REF_WT for L2:H2-L1 REF_WT) H2- identifierMedian H2- L1 Scalar for VAR2 H2- Scalar L2:H2-L1 set H2-L2:H2-L1L2:H2-L1 (Median) VAR2 L2:H2-L1 (Median) Scalar 58-57 110:1_94:1 99:014.6195 E 38:62 −0.4975 5.12  3-19 81:45_60:41 62:38 0.472 E 38:62−0.4975 0.97 108-107 92:3_78:2 97:03 3.546 E 38:62 −0.4975 4.04308*-320* 53:34_63:44 60:40 0.405 E 38:62 −0.4975 0.9 319*-310*110:9_75:13 89:11 2.1055 E 38:62 −0.4975 2.6 26-93 103:1_82:1 99:014.522 E 38:62 −0.4975 5.02 65-72 101:1_79:8 97:03 3.477 E 62:38 0.49752.98 306*-304* 97:1_101:3 98:02 3.981 E 62:38 0.4975 3.48 51-5282:8_96:10 91:09 2.323 E 62:38 0.4975 1.83 307*-305* 90:9_79:17 87:131.9365 E 62:38 0.4975 1.44 58-57 89:1_89:1 99:01 4.488 A 39:61 −0.42954.92  3-19 59:35 63:37 0.527 A 39:61 −0.4295 0.96 108-107 58:19 75:251.122 A 39:61 −0.4295 1.55 309*-310* 87:06 94:06 2.7 A 39:61 −0.42953.13 26-93 103:1_73:1 99:01 4.4615 A 39:61 −0.4295 4.89 65-72 66:1384:16 1.644 A 61:39 0.4295 1.21 306*-304* 103:1_92:1 99:01 4.548 A 61:390.4295 4.12 51-52 73:18 80:20 1.413 A 61:39 0.4295 0.98 307*-305*73:17_73:20 80:20 1.402 A 61:39 0.4295 0.97 53-54 63:15 81:19 1.459 A61:39 0.4295 1.03 70:16_84:21_84:21_(—) 80:20 1.369 N/A N/A N/A N/A72:44_66:50 66-67 115:1_104:1 99:01 4.6925 B 80:20 1.369 3.32 57-58105:1_94:1 99:01 4.5975 B 80:20 1.369 3.23  182-317* 72:12 86:14 1.791 B80:20 1.369 0.42 Unique identifier REF_WT REF_WT set or VAR1 or VAR2Design Type H1_Ab H1_mutation L1_Ab K F N/A D3H44 D3H44 57-58electrostatic D3H44 L143K_D146G D3H44 305*-307* combination (steric +D3H44 V37W D3H44 electrostatic) 93-26 combination (steric + D3H44 Q39RD3H44 electrostatic) 48-47 electrostatic PERT WT PERT 323*-324* stericPERT W103V PERT J G N/A D3H44 D3H44 93-26 combination (steric + PERTQ39R PERT electrostatic) Presence of Unique H-L disulfide identifierbond (C233- set L1_mutation L1_tag H2_Ab H2_mutation L2_Ab L2_mutationL2_tag C214) (y/n) HA PERT PERT FLAG n 57-58 Q124E_V133D HA PERTL143E_K145T PERT Q124R FLAG n 305*-307* F98A HA PERT Q39R PERT Q38E FLAGn 93-26 Q38E_F98W HA PERT V37W_Q39E PERT Q38R_F98A FLAG n 48-47 WT FLAGD3H44 V37E_F100D D3H44 L89R_F98W HA n 323*-324* P44W_Q89W_F98A FLAGD3H44 V37W_F100W D3H44 F98A HA n FLAG PERT PERT HA n 93-26 Q38E_F98W HAD3H44 V37W_Q39E D3H44 Q38R_F98A FLAG n Unique Normalized H1-L1:H1-identifier Observed Trends Median H1- L2 Scalar set for REF_WTsH1-L1:H1-L2 L1:H1-L2 (Median) Preference of H_D3H44 34:65_29:70_(—)29:71 −0.909 for FLAG_L_PERT over 27:67_29:73_(—) HA_L_D3H44 isconsistent 27:73_2:101_1:98 (Inconsistency in observed ratios of H_D3H44towards FLAG_L_D3H44 or HA_L_PERT likely due to HA-tag issue).Preference of H_PERT for L_PERT over L_D3H44. 57-58 90:3_85:4 96:043.1675 305*-307* 102:3_99:3_(—) 96:04 3.2045 101:4_121:5_1 10:10_85:2493-26 76:24_70:31 73:27 0.9895 48-47 112:01 99:01 4.336 323*-324* 71:0199:01 4.267 Inconsistency in observed 92:4_88:8_(—) 53:47 0.119 ratiosof H_D3H44 towards 58:52_43:60_(—) FLAG_L_D3H44 or 47:70 HA_L_PERTlikely due to HA-tag issue, as preference of H_D3H44 for FLAG_L_(—) PERTover HA_L_D3H44 is consistent. Preference of H_PERT for L_PERT overL_D3H44. 93-26 89:1_88:6 97:03 3.5745 Normalized REF_WT for Median VAR1H1- Δ (VAR1- Unique REF_WT for L1:H1-L2 REF_WT) H1- identifier REF_WTVAR1 H1- Scalar L1:H1-L2 set for VAR1 L1:H1-L2 (Median) Scalar N/A N/AN/A N/A 57-58 K 29:71 −0.909 4.08 305*-307* K 29:71 −0.909 4.11 93-26 K29:71 −0.909 1.9 48-47 F 94:06 2.804 1.53 323*-324* F 94:06 2.804 1.46N/A N/A N/A N/A 93-26 G 73:27 0.998 2.58 Normalized REF_WT for MedianVAR2 H2- Δ (VAR2- Unique Normalized H2-L2:H2- REF_WT REF_WT for L2:H2-L1REF_WT) H2- identifier Median H2- L1 Scalar for VAR2 H2- Scalar L2:H2-L1set H2-L2:H2-L1 L2:H2-L1 (Median) VAR2 L2:H2-L1 (Median) Scalar106:1_104:1_123:2_105:6_(—) 94:06 2.804 N/A N/A N/A N/A126:9_102:7_113:15 57-58 112:1_99:10 97:03 3.515 F 94:06 2.804 0.71305*-307* 113:1_97:1_83:1_108:2_(—) 99:01 4.2825 F 94:06 2.804 1.48100:2_115:2 93-26 101:1_84:1 99:01 4.4435 F 94:06 2.804 1.64 48-47 82:0297:03 3.519 K 29:71 −0.909 4.43 323*-324* 92:17 84:16 1.675 K 29:71−0.909 2.58 98:26_68:23_68:25_65:44_(—) 73:27 0.998 N/A N/A N/A N/A67:46 93-26 96:1_69:32 94:06 2.673 J 53:47 0.119 2.55

TABLE 18 Presence of H-L disulfide REF_(—) H1_(—) H1_(—) L1_(—) L2_(—)bond (C233- WT Ab mutation L1_Ab mutation L1_tag L2_Ab mutation L2_tagC214) (y/n) Observed trends E PERT WT PERT WT FLAG PERT WT HA n Apparenttag dependence observed. Observed ratios are likely due to HA-tagcleavage rather than tag interference with pairing A TRAS WT TRAS WTFLAG TRAS WT HA n Apparent tag dependence. Likely similar issue withHA-tag as above, but to a lesser extent O D3H44 WT D3H44 WT HA D3H44 WTFLAG n No apparent tag dependence observed for these ratios H D3H44 WTD3H44 WT FLAG TRAS WT HA n Preference of H_D3H44 for L_TRAS over I D3H44WT D3H44 WT HA TRAS WT FLAG n L_D3H44 J D3H44 WT D3H44 WT FLAG PERT WTHA n Inconsistency in observed ratios of K D3H44 WT D3H44 WT HA PERT WTFLAG n H_D3H44 towards FLAG_L_(—) D3H44 or HA_L_PERT likely due toHA-tag issue, as preference of H_D3H44 for FLAG_L_PERT over HA_L_D3H44is consistent B TRAS WT TRAS WT FLAG D3H44 WT HA n Preference of H_TRASfor L_TRAS over C TRAS WT TRAS WT HA D3H44 WT FLAG n L_D3H44 M TRAS WTTRAS WT FLAG PERT WT HA n Likely no preference of H_TRAS for L_TRAS NTRAS WT TRAS WT HA PERT WT FLAG n over L_PERT F PERT WT PERT WT FLAGD3H44 WT HA n Preference of H_PERT for L_(—) PERT over G PERT WT PERT WTHA D3H44 WT FLAG n L_D3H44 D PERT WT PERT WT FLAG TRAS WT HA n Likely nopreference of H_PERT for L_PERT L PERT WT PERT WT HA TRAS WT FLAG n overL_TRAS H1-L1 Antigen H1-L1 Antigen Normalized Number of H1-L1 Tm H1-L2Tm Affinity-TF Affinity-HER2 Median H1- screening (Range if (Range if(KD) (Range if (KD) (Range if REF_WT H1-L1:H1-L2 L1:H1-L2 experimentsn > 1) (° C.) n > 1) (° C.) n > 1) (nM) n > 1) (nM) E81:20_79:20_70:29_78:34_78:47_57:35_(—) 62:38 10 73.25 73.26 N/A ND56:36_65:53_65:53_53:50 A 66:34_57:36_66:43_54:36_58:43_58:43 61:39 676.93 76.86 N/A 0.696 O 50:55_48:70 44:56 2 75.88 75.77 0.0522 N/A(3.57) (0.08822) H 36:59_36:59_35:86_31:87_20:67 29:71 5 75.77 ND 0.0622N/A (0.73) (0.12039) I 50:58_49:61_34:68_21:71_21:76 33:67 5 75.88 ND0.0522 N/A (3.57) (0.08822) J 92:4_88:8_58:52_43:60_47:70 53:47 5 75.7779.47 0.0622 N/A (0.73) (0.12039) K 34:65_29:70_27:67_29:73_27:73_(—)29:71 7 75.88 79.76 0.0522 N/A 2:101_1:98 (3.57) (0.08822) B70:16_84:21_84:21_72:44_66:50 80:20 5 76.93 ND N/A 0.696 C107:15_107:18_86:17_89:18_61:19 84:16 5 76.86 ND N/A 0.413 M 45:43 51:491 76.93 ND N/A 0.696 N 38:51 42:58 1 76.86 ND N/A 0.413 F106:1_104:1_123:2_105:6_126:9_102:7_(—) 94:06 7 73.25 66.43 N/A ND113:15 G 98:26_68:23_68:25_65:44_67:46 73:27 5 73.26 66.79 N/A ND D68:43_66:42_55:56_56:58_48:60 49:51 5 73.25 ND N/A ND L 42:58 42:58 173.26 ND N/A ND

TABLE 19 Unique Fab identifer Region Design Type H1_mutation L1_mutationL2_mutation 57 constant electrostatic L143K_D146G Q124E_V133D Q124R 182 variable electrostatic V37E_F100D L89R_F98W F98Y 306* constantcombination A139G_K145T_D146G_Q179E_(—) L135W F116A_Q124E_L135A_T180E(steric + V190A electrostatic) 58 constant electrostatic L143E_K145TQ124R Q124E_V133D 107  constant steric A139G_V190A L135W F116A_L135A304* constant combination A139W_S186K F116A_Q124E_L135A_T180E L135W(steric + electrostatic) 93 variable combination Q39R Q38E_F98WQ38R_F98A (steric + electrostatic) 165  constant combination A139WF116A_L135A S131R_L135W (steric + electrostatic) 65 constantelectrostatic K145T_Q179D_S188L Q160K_T178R Q124E_Q160E_T178D 66constant electrostatic L143A_D146G_Q179R Q124E_V133W_Q160E_T180EV133A_Q160K_T178R 313* variable steric V37W F98A P44W 314* constantcombination A139V_K145L_Q179E_S188G_(—) F116A_S131K_V133G_S176F_(—)F118W_V133S_S176A_T180E (steric + V190S T178A electrostatic) 26 variablecombination V37W_Q39E Q38R_F98A Q38E_F98W (steric + electrostatic) 324*variable steric V37W_F100W F98A P44W_Q89W_F98A  3 constant stericF174V_P175S_S188G S176L V133S 72 constant electrostatic D146G_Q179RQ124E_Q160E_T178D Q160K_T178R 307* variable combination Q39R Q38E F98A(steric + electrostatic) 305* variable combination V37W F98A Q38E(steric + electrostatic) 52 variable electrostatic Q39E Q38R Q38E 315*constant combination A139W_S186K_S188A F118W_V133S_S176A_T180EF116A_S131K_V133G_S176F_(—) (steric + T178A electrostatic) 108  constantsteric A139W F116A_L135A L135W 19 constant steric S188L_V190Y V133SS176L 51 variable electrostatic Q39R Q38E Q38R Median Δ (VAR-REF_WT)Number Unique H1-L1:H1- of identifer L2 Scalar H1-L1/L2 Systems Systems57 4.305 TRAS-TRAS/TRAS; TRAS-TRAS/D3H44; PERT-PERT/PERT;PERT-PERT/D3H44; D3H44-D3H44/TRAS; 6 D3H44-D3H44/PERT 182  4.055D3H44-D3H44/TRAS; D3H44-D3H44/PERT 2 306* 3.99 TRAS-TRAS/TRAS;TRAS-TRAS/D3H44; PERT-PERT/PERT; PERT-PERT/D3H44 4 58 3.23TRAS-TRAS/TRAS; TRAS-TRAS/D3H44; PERT-PERT/TRAS; PERT-PERT/PERT;PERT-PERT/D3H44 5 107  3.03 TRAS-TRAS/TRAS; TRAS-TRAS/D3H44;PERT-PERT/PERT; PERT-PERT/D3H44 4 304* 3.03 TRAS-TRAS/TRAS;TRAS-TRAS/D3H44; PERT-PERT/TRAS; PERT-PERT/PERT; PERT-PERT/D3H44 5 932.9 TRAS-TRAS/TRAS; TRAS-TRAS/D3H44; PERT-PERT/PERT; PERT-PERT/D3H44;D3H44-D3H44/PERT 5 165  2.8 TRAS-TRAS/D3H44; PERT-PERT/D3H44;D3H44-D3H44/PERT 3 65 2.6 TRAS-TRAS/TRAS; PERT-PERT/PERT;PERT-PERT/D3H44 3 66 2.565 TRAS-TRAS/D3H44; PERT-PERT/D3H44;D3H44-D3H44/TRAS; D3H44-D3H44/PERT 4 313* 2.55 TRAS-TRAS/D3H44;D3H44-D3H44/PERT 2 314* 2.495 TRAS-TRAS/D3H44; PERT-PERT/D3H44 2 262.285 TRAS-TRAS/TRAS; TRAS-TRAS/D3H44; PERT-PERT/TRAS; PERT-PERT/PERT;PERT-PERT/D3H44; 6 D3H44-D3H44/PERT 324* 2.2 D3H44-D3H44/TRAS;D3H44-D3H44/PERT 2  3 2.13 TRAS-TRAS/TRAS; TRAS-TRAS/D3H44;PERT-PERT/TRAS; PERT-PERT/PERT; PERT-PERT/D3H44 5 72 1.725TRAS-TRAS/TRAS; TRAS-TRAS/D3H44; PERT-PERT/TRAS; PERT-PERT/PERT;PERT-PERT/D3H44; 6 D3H44-D3H44/TRAS 307* 1.71 TRAS-TRAS/TRAS;TRAS-TRAS/D3H44; PERT-PERT/PERT; PERT-PERT/D3H44; D3H44-D3H44/PERT 5305* 1.44 TRAS-TRAS/TRAS; PERT-PERT/PERT; D3H44-D3H44/PERT 3 52 1.405TRAS-TRAS/TRAS; PERT-PERT/TRAS; PERT-PERT/PERT; PERT-PERT/D3H44 4 315*1.19 TRAS-TRAS/D3H44; PERT-PERT/D3H44 2 108  1.15 TRAS-TRAS/TRAS;TRAS-TRAS/D3H44; PERT-PERT/TRAS; PERT-PERT/PERT; PERT-PERT/D3H44 5 190.945 TRAS-TRAS/TRAS; TRAS-TRAS/D3H44; PERT-PERT/PERT; PERT-PERT/D3H44 451 0.935 TRAS-TRAS/TRAS; TRAS-TRAS/D3H44; PERT-PERT/PERT;PERT-PERT/D3H44 4

TABLE 20 Unique identifier set Fab Region Design Type H1_mutationL1_mutation H2_mutation L2_mutation 306*-304* constant combinationA139G_K145T_D146G_(—) L135W A139W_S186K F116A_Q124E_L135A_(—) (steric +Q179E_V190A T180E electrostatic) 307*-305* variable combination Q39RQ38E V37W F98A (steric + electrostatic)  3-19 constant stericF174V_P175S_S188G S176L S188L_V190Y V133S 93-26 variable combinationQ39R Q38E_F98W V37W_Q39E Q38R_F98A (steric + electrostatic) 58-57constant electrostatic L143E_K145T Q124R L143K_D146G Q124E_V133D 107-108constant steric A139G_V190A L135W A139W F116A_L135A 72-65 constantelectrostatic D146G_Q179R Q124E_Q160E_T178D K145T_Q179D_S188LQ160K_T178R 52-51 variable electrostatic Q39E Q38R Q39R Q38E 319*-310*variable electrostatic V37E_F100D Q89R_F98W WT WT Median Δ Median ΔUnique (VAR1- (VAR2-REF_WT) identifier REF_WT)H1- H2-L2:H2-L1 setL1:H1-L2 Scalar H1-L1/H2-L2 Systems Number of Systems Scalar 306*-304*4.66 PERT-PERT/PERT-PERT; TRAS-TRAS/ 2 3.8 TRAS-TRAS 307*-305* 4.57PERT-PERT/D3H44-D3H44; PERT-PERT/ 3 1.44 PERT-PERT; TRAS-TRAS/TRAS-TRAS 3-19 3.84 PERT-PERT/PERT-PERT; TRAS-TRAS/ 2 0.965 TRAS-TRAS 93-26 3.735D3H44-D3H44/PERT-PERT; PERT-PERT/ 4 3.27 D3H44-D3H44;PERT-PERT/PERT-PERT; TRAS-TRAS/TRAS-TRAS 58-57 3.545PERT-PERT/PERT-PERT; TRAS-TRAS/ 4 4.725 TRAS-TRAS;TRAS-TRAS/D3H44-D3H44; PERT-PERT/D3H44-D3H44 107-108 2.795PERT-PERT/PERT-PERT; TRAS-TRAS/ 2 0.755 TRAS-TRAS 72-65 2.095PERT-PERT/PERT-PERT; TRAS-TRAS/ 2 1.78 TRAS-TRAS 52-51 1.405PERT-PERT/PERT-PERT; TRAS-TRAS/ 2 1.845 TRAS-TRAS 319*-310* 2.33PERT-PERT/PERT-PERT; TRAS-TRAS/ 2 2.865 TRAS-TRAS

TABLE 21 Unique H1:L1:L2 identifier Fab Region Design Type H1_mutationL1_mutation L2_mutation DNA Ratio H1-L1_H1-L2 WT WT WT 1:01:0132.5_45.0_38.9_41.5 325* variable steric V37W F98A F98W 1:01:0111.5_11.9_15.2_8.4 31 variable steric WT F98W F98A 1:01:0129.7_38.9_40.9_44.4 52 variable electrostatic Q39E Q38R Q38E 1:01:0119.1_28.5 51 variable electrostatic Q39R Q38E Q38R 1:01:01 25.4_23.7108  constant steric A139W F116A_L135A L135W 1:01:01 12.1_28.4 305*variable combination V37W F98A Q38E 1:01:01 6.6_9.5_8.9 (electrostatic +steric) 307* variable combination Q39R Q38E F98A 1:01:0110.1_9.6_9.9_8.6 (electrostatic + steric) 19 constant steric S188L_V190YV133S S176L 1:01:01 43.2_26.5  3 constant steric F174V_P175S_(—) S176LV133S 1:01:01 42.8_43.5 S188G 72 constant electrostatic D146G_Q179RQ124E_Q160E_(—) Q160K_T178R 1:01:01 7.1_6.3 T178D 65 constantelectrostatic K145T_Q179D_(—) Q160K_T178R Q124E_Q160E_(—) 1:01:0125.3_41.6 S188L T178D Unique median Median median identifier H1-L1_H1-L2H1-L1_H1-L1 H1-L1_H1-L1 H1-L2_H1-L2 H1-L2_H1-L2 40.2 3.7_11.5_4.6_24.18.05 63.8_43.5_55.7_34.4 49.6 325* 11.7 84.1_84.9_82.1_90.3 84.54.4_2.9_2.6_1.2 2.75 31 39.9 66.7_52.9_51.5_42.4 52.2 3.6_8.1_7.6_13.07.85 52 23.8 80.9_52.5 66.7 0.0_18.8 9.4 51 24.55 71.1_71.3 71.2 3.6_4.94.25 108  20.25 87.9_71.4 79.65 0_0 0 305* 8.9 92.6_86.2_88.7 88.70_3.8_2.3 2.3 307* 9.75 89.9_90.3_89.8_91.0 90.1 0_0_0.3_0.3 0.15 1934.85 36.7_72.4 54.55 20.1_0.7 10.4  3 43.15 47.1_47.2 47.15 10.1_8.99.5 72 6.7 92.9_93.7 93.3 0_0 0 65 33.45 71.8_29.0 50.4 3.0_29.3 16.15

TABLE 22 Unique identifier H1- H2- set Fab region Design Type L1_AbH1_mutation L1_mutation L2_Ab H2_mutation D3H44 WT WT PERT WT 305*-307*variable combination D3H44 V37W F98A PERT Q39R (electrostatic + steric)154-152 variable combination D3H44 V37W_Q39E Q38R_F98A PERTV37A_Q39R_W103V (electrostatic + steric) 326*-23  constant electrostaticD3H44 S186R Q124E_Q160E_T180E PERT K145L_Q179E 327*-328* combinationcombination D3H44 Q39E_S186R Q38R_Q124E_Q160E_(—) PERT Q39R_K145L_Q179Eof constant (electrostatic) T180E and variable 329*-330* constantcombination *D3H44 A139G_V190A L135W PERT A139W_K145Y_Q179E(electrostatic + steric) 331*-257  constant electrostatic *D3H44D146G_Q179K Q124E_Q160E_T180E PERT L143E_K145T 329*-330* constantcombination *D3H44 A139G_V190A L135W PERT A139W_K145Y_Q179E(electrostatic + steric) 332*-284  constant electrostatic D3H44D146G_Q179K Q124E_Q160E_T180E PERT L143E_K145T 333*-334* combinationcombination D3H44 Q39E_S186R Q38R_Q160E_T180E PERT Q39R_K145T_Q179E ofconstant (electrostatic) and variable 335*-336* combination combinationD3H44 V37W_L124E F98A_V133A_S176K PERT L124R of constant(electrostatic + and variable steric) 331*-257  constant electrostatic*D3H44 D146G_Q179K Q124E_Q160E_T180E PERT L143E_K145T Unique SEC stepidentifier H1:H2:L1:L2 performed Number of paired:mispairedpaired:mispaired set L2_mutation DNA ratio post pA? experimentsspecies(all) species (mean) WT 22:8:53:17 Y 2 3:97_2:98   2:98 305*-307*Q38E 24:6:56:14 Y 1 100:0 100:0 154-152 Q38E_P44W 15:15:35:35 N 1 100:0100:0 326*-23  S131K 22:8:53:17 N 1 100:0 100:0 327*-328* Q38E_S131K22:8:46:24 N 1 100:0 100:0 329*-330* F116A_S131K_L135A 15:15:35:35 N 1100:0 100:0 331*-257  Q124R_Q160K_T178R 22:8:35:35 N 1 100:0 100:0329*-330* F116A_S131K_L135A 15:15:35:35 N 1 100:0 100:0 332*-284  Q124R22:8:53:17 N 1  98:2  98:2 333*-334* Q38E_S131K 22:8:46:24 N 1  98:2 98:2 335*-336* F98W_V133G_S176D 22:8:46:24 N 1  98:2  98:2 331*-257 Q124R_Q160K_T178R 22:8:46:24 N 1  97:3  97:3 H1-L1_H2- H1-L1_H2-L2Unique Δ (VAR-REF_WT) L2 (and (and H1- H1- H1- H1- H2- identifierpaired_over_(—) paired_over_(—) H1-L2_H2- L2_H2-L1) L1_H1- L1_H1- L2_H1-L1_H2- set mispaired_Scalar mispaired_Scalar L1) side peak L1 L2 L2 L1−3.72 0 1.55 not present 0 0 0 0 305*-307* 5 8.72 95 10 0 0 0 0 154-1525 8.72 94.4 6.8 0 0 0 0 326*-23  5 8.72 80.8 4.2 1.9 0 0 0 327*-328* 58.72 70.7 6.5 1.2 0 0 0 329*-330* 5 8.72 61.9 3.1 0 0 0 0 331*-257  58.72 56.4 3.4 6.1 0 0 0 329*-330* 5 8.72 42.2 3.2 0 0 0 0 332*-284  4.067.78 32.5 1.8 11.5 0 0 0 333*-334* 4 7.72 85.9 7.2 0 0 0 0 335*-336*3.84 7.56 66.4 6.7 0 0 0 0 331*-257  3.51 7.23 50.5 3.4 2.8 0 0 0 Uniqueidentifier H2-L1_H2- H2-L2_H2- H1-L1_H2- H1-L2_H2- set L2 L2 L1 L2 H1-L1H1-L2 H2-L1 H2-L2 0 0 0 96.9 0 0.7 0 0.9 305*-307* 0 2.8 0 0 2.2 0 0 0154-152 0 0 0 0 3.1 0 0 2.5 326*-23  0 0 0 0 14 0 0 3.3 327*-328* 0 0 00 26.6 0 0 1.5 329*-330* 0 1.1 0 0 2.6 0 0 34.3 331*-257  0 0 0 0 33.9 00 3.6 329*-330* 0 2.4 0 0 0 0 0 55.4 332*-284  0 0 1.7 0 54.3 0 0 0333*-334* 0 0 0 1.8 9.3 0 0 3 335*-336* 0 4.2 0 0 25.4 2.1 0 2 331*-257 2.9 0 0 0 42.6 0 0 1.3 Unique identifier H1- H2- set Fab region DesignType L1_Ab H1_mutation L1_mutation L2_Ab H2_mutation 90-92 variablecombination D3H44 V37W_Q39E Q38R_F98A PERT V37I_Q39R (electrostatic +steric) 34-39 variable combination D3H44 V37W_Q39E Q38R_F98A PERT Q39R(electrostatic + steric) 313*-337* variable steric D3H44 V37W F98A PERTL45A 336*-335* combination combination D3H44 L124R F98W_V133G_S176D PERTV37W_L124E of constant (electrostatic + and variable steric) 338*-299 variable steric D3H44 V37W_W103F F98A PERT L45A 313*-339* variablesteric D3H44 V37W F98A PERT W103V 340*-337* variable steric D3H44V37W_W103F F98A PERT L45A 340*-339* variable steric D3H44 V37W_W103FF98A PERT W103V 66-67 constant electrostatic D3H44 L143A_D146G_Q179RQ124E_V133W_Q160E_(—) PERT K145T_Q179D_S188F T180E 57-58 constantelectrostatic *D3H44 L143K_D146G Q124E_V133D PERT L143E_K145T 341*-342*combination combination D3H44 V37W_K145T_Q179E F98A_S131K PERT S186R ofconstant (electrostatic + and variable steric) 92-90 variablecombination *D3H44 V37I_Q39R Q38D_F98W PERT V37W_Q39E (electrostatic +steric) 325*-31  variable steric D3H44 V37W F98A PERT WT 92-90 variablecombination *D3H44 V37I_Q39R Q38D_F98W PERT V37W_Q39E (electrostatic +steric)  300-343* variable steric D3H44 V37W F98A PERT W103V 342*-341*combination combination D3H44 S186R F98W_Q160E_T180E PERTV37W_K145T_Q179E of constant (electrostatic + and variable steric)344*-121  variable combination D3H44 F100W_W103F F98L PERT Q39R(electrostatic + steric) Unique SEC step identifier H1:H2:L1:L2performed Number of paired:mispaired paired:mispaired set L2_mutationDNA ratio post pA? experiments species(all) species (mean) 90-92Q38D_F98W 15:15:35:35 N 1 96:4 96:4 34-39 Q38E 15:15:35:35 N 1 96:4 96:4313*-337* P44W 15:15:35:35 N 1 95:5 95:5 336*-335* F98A_V133A_S176K22:8:46:24 N 1 94:6 94:6 338*-299  P44F 22:8:46:24 N 1 94:6 94:6313*-339* P44W 22:8:46:24 N 1 93:7 93:7 340*-337* P44W 15:15:35:35 N 192:8 92:8 340*-339* P44W 26:4:56:14 N 1 92:8 92:8 66-67V133A_Q160K_T178R 22:8:53:17 N 1 92:8 92:8 57-58 Q124R 22:8:53:17 N 288:12_93:7 91:9 341*-342* F98W_Q160E_T180E 22:8:46:24 N 1 90:10  90:1092-90 Q38R_F98A 20:10:40:30 N 1 89:11  89:11 325*-31  F98W 8:22:53:17 Y1 88:12  88:12 92-90 Q38R_F98A 22:8:35:35 N 1 87:13  87:13  300-343*P44F 22:8:46:24 N 1 87:13  87:13 342*-341* F98A_S131K 22:8:46:24 N 187:13  87:13 344*-121  Q38E_F98W 22:8:46:24 N 1 86:14  86:14 Unique Δ(VAR-REF_WT) H1-L1_H2-L2 H1-L1_H2-L2 H1- H1- H1- H2- identifierpaired_over_(—) paired_over_(—) (and H1- (and H1-L2_H2- L1_H1- L1_H1-L2_H1- L1_H2- set mispaired_Scalar mispaired_Scalar L2_H2-L1) L1) sidepeak L1 L2 L2 L1 90-92 3.2 6.92 90.6 4.3 0 0 0 0 34-39 3.18 6.9 89.6 3.90 0 0 0 313*-337* 2.99 6.71 82.1 9.6 0 0 0 0 336*-335* 2.68 6.4 64.910.4 0 0 1.5 0 338*-299  2.67 6.39 85.5 4.7 0 0 0 0 313*-339* 2.6 6.3282.1 7.9 0 0 0 0 340*-337* 2.48 6.2 77.5 7.8 0 0 0 0 340*-339* 2.47 6.1912.2 1.6 3.2 0 0 0 66-67 2.42 6.14 26.6 2.2 3 0 0 0 57-58 2.31 6.03 56.43.05 1.8 0 0 0 341*-342* 2.21 5.93 29.8 2.7 8.5 1.6 0 0 92-90 2.13 5.8584.4 6.5 0 0 0 0 325*-31  1.96 5.68 81.8 not annotated 0 0 0 0 92-901.92 5.64 80 6 0 0 0 0  300-343* 1.87 5.59 71.4 4.5 0 0 0 0 342*-341*1.87 5.59 52.1 3.8 2.4 0 0 0 344*-121  1.83 5.55 68.8 7.1 1.7 0 0 0Unique H2- H2- H1- H1- identifier L1_H2- L2_H2- L1_H2- L2_H2- set L2 L2L1 L2 H1-L1 H1-L2 H2-L1 H2-L2 90-92 0 0 0 3.9 2.3 0 0 3.2 34-39 0 0 0 42.8 0 0 3.7 313*-337* 2.1 1.3 0 2.7 1.1 0 0 10.7 336*-335* 1.1 9 3.8 018.6 0 0 1.1 338*-299  0 0 0 6.5 3.6 0 0 4.5 313*-339* 0 1.5 5.1 1.8 5.10 0 4.4 340*-337* 0 0 0 7.7 1.4 0 0 13.4 340*-339* 0 1.9 2 0 74.9 5.8 00 66-67 0 0 2.2 0 62.2 6 0 0 57-58 3.15 1.15 0 5.15 27.35 1.1 0 3.9341*-342* 0 0 0 2.2 51.8 6.1 0 0 92-90 0 0 8.8 1.8 2.3 0 0 2.7 325*-31 0 1.9 6.3 5 0 0 1.1 4 92-90 2.9 1.2 3.1 6.8 4.1 0 0 1.9  300-343* 0 01.8 9.2 9.2 2.4 0 6 342*-341* 0 0 13.3 0 32.2 0 0 0 344*-121  0 0 2.77.1 13.9 4 0 1.8 Unique identifier H1- H2- set Fab region Design TypeL1_Ab H1_mutation L1_mutation L2_Ab H2_mutation 345*-346* combinationcombination D3H44 V37W F98A_L135W PERT A139W of constant (steric) andvariable 347*-348* combination combination D3H44 V37W_K145T_Q179EF98A_S131K PERT WT of constant (electrostatic + and variable steric)57-58 constant electrostatic *D3H44 L143K_D146G Q124E_V133D PERTL143E_K145T 338*-349* variable steric D3H44 V37W_W103F F98A PERT W103F 300-349* variable steric D3H44 V37W F98A PERT W103F 106-97  variablecombination D3H44 V37I_Q39D Q38R_F98W PERT V37W_Q39R_W103F(electrostatic + steric) 111-112 combination combination D3H44Q39D_A139G_V190A Q38R_L135W PERT Q39R_A139W of constant (electrostatic +and variable steric) 350*-31  variable steric D3H44 V37W_W103F F98A PERTWT 338*-343* variable steric D3H44 V37W_W103F F98A PERT W103V 73-74variable combination D3H44 V37I_Q39R Q38E_F98W PERT V37W_Q39E_W103F(electrostatic + steric) 300-299 variable steric D3H44 V37W F98A PERTL45A 152-154 variable combination D3H44 V37A_Q39R_W103V Q38E_P44W PERTV37W_Q39E (electrostatic + steric) 306*-304* constant combination *D3H44A139G_K145T_D146G_(—) L135W PERT A139W_S186K (electrostatic +Q179E_V190A steric) 107-108 constant steric D3H44 A139G_V190A L135W PERTA139W 307*-305* variable combination D3H44 Q39R Q38E PERT V37W(electrostatic + steric) Unique SEC step identifier H1:H2:L1:L2performed Number of paired:mispaired paired:mispaired set L2_mutationDNA ratio post pA? experiments species(all) species (mean) 345*-346*F98W_F116A 22:8:46:24 N 1 86:14 86:14 347*-348* F98W 22:8:46:24 N 185:15 85:15 57-58 Q124R 22:8:53:17 N 1 84:16 84:16 338*-349* P44F22:8:46:24 N 1 83:17 83:17  300-349* P44F 22:8:46:24 N 1 82:18 82:18106-97  Q38E_F98L 22:8:40:30 N 1 81:19 81:19 111-112 Q38D_F116A_L135A22:8:53:17 N 1 80:20 80:20 350*-31  F98W 22:8:46:24 N 1 80:20 80:20338*-343* P44F 22:8:46:24 N 1 80:20 80:20 73-74 Q38R_F98L 22:8:46:24 N 177:23 77:23 300-299 P44F 22:8:46:24 N 1 76:24 76:24 152-154 Q38R_F98A22:8:53:17 N 1 72:28 72:28 306*-304* F116A_Q124E_L135A_(—) 22:8:53:17 N1 71:29 71:29 T180E 107-108 F116A_L135A 15:15:17:53 Y 1 70:30 70:30307*-305* F98A 22:8:46:24 N 1 70:30 70:30 Δ (VAR- Unique REF_WT)H1-L1_H2-L2 H1-L1_H2-L2 H1- H1- H1- H2- identifier paired_over_(—)paired_over_(—) (and H1- (and H1-L2_H2- L1_H1- L1_H1- L2_H1- L1_H2- setmispaired_Scalar mispaired_Scalar L2_H2-L1) L1) side peak L1 L2 L2 L1345*-346* 1.79 5.51 66.9 7.7 0 0 0 0 347*-348* 1.71 5.43 32.9 3.7 6.92.2 0 0 57-58 1.64 5.36 68.7 5 0 0 0 0 338*-349* 1.61 5.33 57.5 5.1 0 00 0  300-349* 1.53 5.25 75.4 6.6 0 0 0 0 106-97  1.45 5.17 44.2 12.8 2.10 0 0 111-112 1.37 5.09 73.6 1.6 0 0 0 0 350*-31  1.37 5.09 66.8 4.6 01.7 1 0 338*-343* 1.36 5.08 53.6 3 1.5 0 0 0 73-74 1.18 4.9 26.6 1.6 1.30 0 0 300-299 1.17 4.89 29.8 2.3 8.3 4.2 0 0 152-154 0.92 4.64 57.9 3.50 0 0 0 306*-304* 0.87 4.59 56.1 0.8 0 0 0 0 107-108 0.86 4.58 0 notpresent 0 0 0 0 307*-305* 0.85 4.57 56.7 8 0 0 0 0 Unique H2- H2- H1-H1- identifier L1_H2- L2_H2- L1_H2- L2_H2- set L2 L2 L1 L2 H1-L1 H1-L2H2-L1 H2-L2 345*-346* 0 0 1.6 7.7 15.4 5 0 3.5 347*-348* 0 2.5 0 3.638.4 9.5 0 4.1 57-58 0 0 0 14.1 12.1 2.1 0 3 338*-349* 0 1.3 4.2 7.6 0 04.8 24.6  300-349* 2.2 0 5 10.6 2.3 0 0 4.4 106-97  0 0 13 0 34.7 6 0 0111-112 0 0 11.8 7.3 0 0 1.2 6.1 350*-31  0 0 11.2 4.4 12.9 1.9 0 0338*-343* 1.4 0 1.3 8.8 22.2 9 0 2.2 73-74 0 0 3.3 2.7 48.6 17.6 0 0300-299 1.4 0 0 5.7 38.2 12.4 0 0 152-154 0 0 5.9 18.9 10.2 3.7 0 3.4306*-304* 0 1.1 7.8 19.1 13.3 2.5 0 0 107-108 0 3.9 0 29.8 0 0 0 66.3307*-305* 0 0 6.5 19.1 8.8 4.4 0 4.5 Unique identifier H1- H2- set Fabregion Design Type L1_Ab H1_mutation L1_mutation L2_Ab H2_mutation351*-312* variable steric D3H44 V37W F98A PERT WT  186-352* variablesteric D3H44 WT F98W PERT V37F 353*-354* constant combination D3H44S186R L135W_Q160E_T180E PERT A139W_K145T_Q179E (electrostatic + steric)1-2 constant electrostatic D3H44 S186R Q124E_Q160E_T178D PERTK145L_Q179E  121-344* variable combination D3H44 Q39R Q38E_F98W PERTF100W_W103F (electrostatic + steric) 72-65 constant electrostatic D3H44D146G_Q179R Q124E_Q160E_T178D PERT K145T_Q179D_S188L 312*-351* variablesteric D3H44 WT WT PERT V37W  186-355* variable steric D3H44 WT F98WPERT F100W_W103F 356*-357* variable steric D3H44 V37F F98L PERT W103F346*-345* combination of combination D3H44 A139W F98W_F116A PERT V37Wconstant and (steric) variable 343*-338* variable steric D3H44 W103VP44F PERT V37W_W103F 52-51 variable electrostatic D3H44 Q39E Q38R PERTQ39R 358*-359* variable steric D3H44 L45A P44F PERT V37F 348*-347*combination of combination D3H44 WT F98W PERT V37W_K145T_Q179E constantand (electrostatic + variable steric) 355*-186  variable steric D3H44F100W_W103F F98L PERT WT 360*-359* variable steric D3H44 W103V P44F PERTV37F 313*-361* variable steric D3H44 V37W F98A PERT W103F 340*-361*variable steric D3H44 V37W_W103F F98A PERT W103F 357*-356* variablesteric D3H44 W103F P44W PERT V37F 359*-362* variable steric D3H44 V37FF98L PERT W103F  186-363* variable steric D3H44 WT F98W PERT V37F_W103F363*-186  variable steric D3H44 V37F_W103F F98L PERT WT 299-300 variablesteric D3H44 L45A P44F PERT V37W 359*-358* variable steric D3H44 V37FF98L PERT L45A 356*-364* variable steric D3H44 V37F F98L PERT L45AUnique SEC step identifier H1:H2:L1:L2 performed Number ofpaired:mispaired paired:mispaired set L2_mutation DNA ratio post pA?experiments species(all) species (mean) 351*-312* WT 22:8:46:24 N 169:31 69:31  186-352* F98L 22:8:46:24 N 1 66:34 66:34 353*-354*F116A_S131K 22:8:46:24 N 2 73:27_60:40 66:34 1-2 S131K 22:8:53:17 Y 165:35 65:35  121-344* F98L 22:8:46:24 N 1 65:35 65:35 72-65 Q160K_T178R15:15:53:17 Y 1 63:37 63:37 312*-351* F98A 22:8:46:24 N 1 62:38 62:38 186-355* F98L 22:8:46:24 N 1 59:41 59:41 356*-357* P44W 22:8:46:24 N 158:42 58:42 346*-345* F98A_L135W 22:8:46:24 N 1 58:42 58:42 343*-338*F98A 22:8:46:24 N 1 58:42 58:42 52-51 Q38E 8:22:53:17 Y 1 58:42 58:42358*-359* F98L 22:8:46:24 N 1 58:42 58:42 348*-347* F98A_S131K22:8:46:24 N 1 56:44 56:44 355*-186  F98W 22:8:46:24 N 1 56:44 56:44360*-359* F98L 22:8:46:24 N 1 56:44 56:44 313*-361* P44W 22:8:46:24 N 155:45 55:45 340*-361* P44W 22:8:46:24 N 1 51:49 51:49 357*-356* F98L22:8:46:24 N 1 50:50 50:50 359*-362* P44F 22:8:46:24 N 1 50:50 50:50 186-363* F98L 22:8:46:24 N 1 49:51 49:51 363*-186  F98W 22:8:46:24 N 149:51 49:51 299-300 F98A 22:8:46:24 N 1 48:52 48:52 359*-358* P44F22:8:46:24 N 1 48:52 48:52 356*-364* P44W 22:8:46:24 N 1 48:52 48:52 Δ(VAR- Unique REF_WT) H1-L1_H2- H1-L1_H2-L2 H1- H1- H1- H2- identifierpaired_over_(—) paired_over_(—) L2 (and H1- (and H1-L2_H2- L1_H1- L1_H1-L2_H1- L1_H2- set mispaired_Scalar mispaired_Scalar L2_H2-L1) L1) sidepeak L1 L2 L2 L1 351*-312* 0.79 4.51 62 4.9 0 0 0 0  186-352* 0.68 4.463.3 5.4 0 0 0 0 353*-354* 0.675 4.39 44.15 4.3 0.75 0.8 0 0 1-2 0.644.36 55.5 not annotated 1.2 0 1.3 0  121-344* 0.62 4.34 54.8 4.5 0 0 0 072-65 0.55 4.27 57.5 not present 0 0 0 0 312*-351* 0.51 4.23 48.8 4.4 00 0 0  186-355* 0.38 4.1 59.4 2 0 0 0 0 356*-357* 0.32 4.04 51.4 7.3 0 00 0 346*-345* 0.32 4.04 44.8 4.5 0 0 0 0 343*-338* 0.32 4.04 42.3 3.8 00 0 0 52-51 0.32 4.04 38.6 not present 0 0 0 0 358*-359* 0.3 4.02 46.43.5 0 0 0 0 348*-347* 0.26 3.98 45 4.4 0 1.3 0 0 355*-186  0.24 3.9610.3 0.8 3.5 3.1 1.5 0 360*-359* 0.23 3.95 44.7 3.9 0 0 0 0 313*-361*0.21 3.93 47.3 5.6 0 0 0 1.6 340*-361* 0.04 3.76 40.8 5.3 0 0 0 0357*-356* 0.02 3.74 46.3 3.1 0 0 0 0 359*-362* 0.01 3.73 38.9 2.3 0 0 00  186-363* −0.03 3.69 36.2 1.3 0 0 1 0 363*-186  −0.03 3.69 33.2 3 0 00 0 299-300 −0.07 3.65 39.2 4.5 0 0 0 0 359*-358* −0.08 3.64 39.7 3.6 00 0 0 356*-364* −0.1 3.62 42 5.5 0 0 0 0 Unique H2- H2- H1- H1-identifier L1_H2- L2_H2- L1_H2- L2_H2- set L2 L2 L1 L2 H1-L1 H1-L2 H2-L1H2-L2 351*-312* 0 1.1 2.1 27.3 2.3 1.8 0 3.4  186-352* 0 0 14.1 9.8 1.83.8 6 1.3 353*-354* 0 0.5 0 30.4 8.85 2.75 0 11.75 1-2 1.9 1.1 2.7 28.72 0 0 5.6  121-344* 0 0 15 14.6 7 3.5 1.8 3.4 72-65 0 0 5.4 30 2.1 0 1.13.8 312*-351* 0 1.3 10 20.2 4.6 3.5 3.8 7.7  186-355* 0 0 10.9 29.7 0 00 0 356*-357* 0 0 8.1 31.7 1.8 2.2 0 4.9 346*-345* 1 0 9.2 25.5 10 6.2 03.1 343*-338* 1.7 3.4 14 20.1 7.7 4.1 2.2 4.4 52-51 1.1 14 7.1 33.9 0 00 5.3 358*-359* 1.4 2.1 20.3 13.5 4.7 3.5 3.8 4.3 348*-347* 0 0 27.9 7.82.4 0 6.6 9 355*-186  0 0 1.3 7.4 42.2 30.7 0 0 360*-359* 0 0 29 7.9 94.2 3.1 2.1 313*-361* 2.1 0 36.3 1.8 5.2 0 3.1 2.7 340*-361* 0 0 43.11.7 7.2 1.3 2.9 3.1 357*-356* 0 0 1.7 37.6 1.3 7 3.2 2.9 359*-362* 0 01.3 37.2 11.4 11.2 0 0  186-363* 0 0 9.6 8.6 13 31.6 0 0 363*-186  0 02.1 32.5 16.1 16.1 0 0 299-300 0 0 14.9 29.8 3.2 3.6 3.3 5.9 359*-358* 00 0 48.3 3.2 3.7 0 5.1 356*-364* 0 0 0 49 2.3 3.5 0 3.2 Uniqueidentifier H1- H2- set Fab region Design Type L1_Ab H1_mutationL1_mutation L2_Ab H2_mutation D3H44 WT WT PERT WT 337*-340* variablesteric D3H44 L45A P44W PERT V37W_W103F 339*-340* variable steric D3H44W103V P44W PERT V37W_W103F 337*-313* variable steric D3H44 L45A P44WPERT V37W 364*-356* variable steric D3H44 L45A P44W PERT V37F 339*-313*variable steric D3H44 W103V P44W PERT V37W 314*-315* constantcombination D3H44 A139V_K145L_Q179E_(—) F116A_S131K_V133G_(—) PERTA139W_S186K_S188A (electrostatic + S188G_V190S S176F_T178A steric) 31-325* variable steric D3H44 WT F98W PERT V37W 349*-300  variablesteric D3H44 W103F P44F PERT V37W 356*-365* variable steric D3H44 V37FF98L PERT W103V  31-350* variable steric D3H44 WT F98W PERT V37W_W103F 257-331* constant electrostatic D3H44 L143E_K145T Q124R_Q160K_T178RPERT D146G_Q179K 19-3  constant steric D3H44 S188L_V190Y V133S PERTF174V_P175S_S188G 352*-186  variable steric D3H44 V37F F98L PERT WT366*-367* constant electrostatic D3H44 S186R Q124E_Q160E_T178D PERTL143E_K145T 343*-300  variable steric D3H44 W103V P44F PERT V37W UniqueSEC step identifier H1:H2:L1:L2 performed Number of paired:mispairedpaired:mispaired set L2_mutation DNA ratio post pA? experimentsspecies(all) species (mean) WT 22:8:53:17 Y 2 3:97_2:98  2:98 337*-340*F98A 22:8:46:24 N 1 47:53 47:53 339*-340* F98A 22:8:46:24 N 1 47:5347:53 337*-313* F98A 22:8:46:24 N 1 46:54 46:54 364*-356* F98L22:8:46:24 N 1 46:54 46:54 339*-313* F98A 22:8:46:24 N 1 46:54 46:54314*-315* F118W_V133S_S176A_(—) 22:8:53:17 N 1 44:56 44:56 T180E 31-325* F98A 22:8:46:24 N 1 42:58 42:58 349*-300  F98A 22:8:46:24 N 141:59 41:59 356*-365* P44W 22:8:46:24 N 1 41:59 41:59  31-350* F98A22:8:46:24 N 1 40:60 40:60  257-331* Q124E_Q160E_T180E 22:8:56:14 Y 139:61 39:61 19-3  S176L 8:22:53:17 Y 1 38:62 38:62 352*-186  F98W22:8:46:24 N 1 37:63 37:63 366*-367* Q124R 22:8:53:17 Y 1 36:64 36:64343*-300  F98A 22:8:46:24 N 1 35:65 35:65 Δ (VAR- H1- Unique REF_WT)L1_H2-L2 H1-L1_H2-L2 H1- H1- H1- H2- identifier paired_over_(—)paired_over_(—) (and H1- (and H1-L2_H2- L1_H1- L1_H1- L2_H1- L1_H2- setmispaired_Scalar mispaired_Scalar L2_H2-L1) L1) side peak L1 L2 L2 L1−3.72 0 1.55 not present 0 0 0 0 337*-340* −0.13 3.59 41.3 4.1 0 0 0 0339*-340* −0.13 3.59 40.3 2.8 0 0 0 0 337*-313* −0.15 3.57 40.6 3.4 0 00 0 364*-356* −0.15 3.57 37.1 3 0 0 0 0 339*-313* −0.18 3.54 33.7 2.2 00 0 0 314*-315* −0.23 3.49 9.5 0.2 1.7 0 0 0  31-325* −0.34 3.38 33.62.6 0 0 0 0 349*-300  −0.35 3.37 37.3 2.2 0 0 0 0 356*-365* −0.36 3.3625.7 3.4 0 0 1 0  31-350* −0.42 3.3 30.7 3.2 0 0 0 0  257-331* −0.443.28 30.2 6.3 0 0 2.1 0 19-3  −0.48 3.24 16.8 not present 2.6 4.1 0 0352*-186  −0.52 3.2 27.5 3.2 0 0 4.1 0 366*-367* −0.57 3.15 26.9 notannotated 1.8 0 0 0 343*-300  −0.61 3.11 20 1.4 0 0 0 0 Unique H2- H2-H1- H1- identifier L1_H2- L2_H2- L1_H2- L2_H2- set L2 L2 L1 L2 H1-L1H1-L2 H2-L1 H2-L2 0 0 0 96.9 0 0.7 0 0.9 337*-340* 0 0 11.7 36.3 5.4 5.30 0 339*-340* 0 0 14.5 31.1 4.8 5.7 1.9 1.7 337*-313* 0 0 11.5 36.4 1.53.1 2.8 4 364*-356* 0 0 21.9 23.5 4.4 4.3 4.1 4.7 339*-313* 0 1.4 5.835.7 8.2 11.6 1.4 2.2 314*-315* 0 0 6.2 13.2 32 36.3 0 1.2  31-325* 0 06.7 42.2 4.5 6.4 3.1 3.4 349*-300  0 0 2.6 45.5 3 10.5 0 1.1 356*-365*1.1 1.7 1.2 29.7 12.1 26 0 1.6  31-350* 0 0 6.1 43.3 4.1 8.9 2.1 4.8 257-331* 1.1 1.6 0 54.7 1.9 3.1 0 5.4 19-3  0 6.1 2.5 55.1 0 0 0 12.8352*-186  0 0 1.1 41 9.8 16.6 0 0 366*-367* 0 2 2.7 59 1.6 2.1 0 3.9343*-300  0 0 1.1 35.1 15.3 28.5 0 0 Unique identifier H1- H2- set Fabregion Design Type L1_Ab H1_mutation L1_mutation L2_Ab H2_mutation D3H44WT WT PERT WT 328*-327* combination of combination D3H44Q39R_K145L_Q179E Q38E_S131K PERT Q39E_S186R constant and (electrostatic)variable 361*-313* variable steric D3H44 W103F P44W PERT V37W 361*-340*variable steric D3H44 W103F P44W PERT V37W_W103F  299-338* variablesteric D3H44 L45A P44F PERT V37W_W103F 368*-369* constant electrostaticD3H44 L143E_K145T_S188L Q124R PERT L143K 370*-371* combination ofcombination D3H44 Q39R_A139W Q38E_F116A_L135V PERT Q39E_A139G_V190Aconstant and (electrostatic + variable steric) 359*-360* variable stericD3H44 V37F F98L PERT W103V 368*-372* constant electrostatic D3H44L143E_K145T_S188L Q124R PERT L143R 373*-374* variable steric D3H44 L45FWT PERT L45A 295-294 constant electrostatic D3H44 L124R V133G_S176D PERTL124E 375*-376* constant electrostatic D3H44 L143E_K145T_S188L Q124RPERT L143R 377*-378* variable steric D3H44 L45A P44F PERT L45F 74-73variable combination D3H44 V37W_Q39E_W103F Q38R_F98L PERT V37I_Q39R(electrostatic + steric) 379*-380* constant electrostatic D3H44L143E_K145T_Q179E Q124K_T178R PERT S186R 381*-382* combination ofcombination D3H44 Q39R_A139W Q38E_F116A PERT Q39E constant and(electrostatic + variable steric) 383*-384* constant electrostatic D3H44H172R WT PERT H172T 349*-338* variable steric D3H44 W103F P44F PERTV37W_W103F 334*-333* combination of combination D3H44 Q39R_K145T_Q179EQ38E_S131K PERT Q39E_S186R constant and (electrostatic) variable378*-377* variable steric D3H44 L45F WT PERT L45A 375*-385* constantelectrostatic D3H44 L143E_K145T_S188L Q124R PERT L143K Unique SEC stepidentifier H1:H2:L1:L2 performed Number of paired:mispairedpaired:mispaired set L2_mutation DNA ratio post pA? experimentsspecies(all) species (mean) WT 22:8:53:17 Y 2 3:97_2:98  2:98 328*-327*Q38R_Q124E_Q160E_T180E 22:8:46:24 N 1 35:65 35:65 361*-313* F98A22:8:46:24 N 1 33:67 33:67 361*-340* F98A 22:8:46:24 N 1 32:68 32:68 299-338* F98A 22:8:46:24 N 1 32:68 32:68 368*-369* V133E 22:8:46:24 N 131:69 31:69 370*-371* Q38R_L135W 22:8:46:24 N 1 31:69 31:69 359*-360*P44F 22:8:46:24 N 1 31:69 31:69 368*-372* V133E 22:8:46:24 N 1 31:6931:69 373*-374* P44W 22:8:46:24 N 1 30:70 30:70 295-294 V133A_S176K22:8:46:24 N 1 30:70 30:70 375*-376* Q124E_V133E 22:8:46:24 N 1 29:7129:71 377*-378* WT 22:8:46:24 N 1 29:71 29:71 74-73 Q38E_F98W 22:8:46:24N 1 28:72 28:72 379*-380* Q124E_T178E_T180E 22:8:46:24 N 1 28:72 28:72381*-382* Q38R_L135W 22:8:46:24 N 1 26:74 26:74 383*-384* N137K_S174R22:8:46:24 N 1 25:75 25:75 349*-338* F98A 22:8:46:24 N 1 24:76 24:76334*-333* Q38R_Q160E_T180E 22:8:46:24 N 1 23:77 23:77 378*-377* P44F22:8:46:24 N 2 21:79_24:76 22:78 375*-385* Q124E_V133E 22:8:46:24 N 121:79 21:79 Δ (VAR- Unique REF_WT) H1-L1_H2- H1-L1_H2-L2 H1- H1- H1- H2-identifier paired_over_(—) paired_over_(—) L2 (and H1- (and H1-L2_H2-L1_H1- L1_H1- L2_H1- L1_H2- set mispaired_Scalar mispaired_ScalarL2_H2-L1) L1) side peak L1 L2 L2 L1 −3.72 0 1.55 not present 0 0 0 0328*-327* −0.63 3.09 5.3 1.1 2 6.5 6.9 0 361*-313* −0.72 3 30.5 2 0 0 00 361*-340* −0.74 2.98 29.2 1.6 0 0 0 0  299-338* −0.77 2.95 27 1.4 0 00 0 368*-369* −0.8 2.92 18.4 2.9 0 0 1.3 0 370*-371* −0.8 2.92 17.9 1.40 1.1 2.4 0 359*-360* −0.81 2.91 17.7 2.1 1.1 0 1.5 0 368*-372* −0.812.91 14.3 1.7 0 1.2 3.6 0 373*-374* −0.83 2.89 20 2.6 0 1.1 1.3 0295-294 −0.85 2.87 13.2 2.6 0 0 2.5 0 375*-376* −0.89 2.83 15.7 2.9 0 01.9 0 377*-378* −0.9 2.82 6.6 0.8 0 0 2.2 0 74-73 −0.92 2.8 5.1 4.6 7.70 4.9 0 379*-380* −0.96 2.76 7.6 1.4 0 2.8 5.4 0 381*-382* −1.06 2.6618.5 1.3 0 0 0 0 383*-384* −1.09 2.63 13.7 1.8 1.2 0 0 0 349*-338* −1.142.58 20.7 0.9 0 0 0 0 334*-333* −1.19 2.53 1 0.3 0 3 5.4 0 378*-377*−1.245 2.48 12.05 1.55 0.55 0 1.4 0 375*-385* −1.3 2.42 11.1 2.4 0 0 0 0Unique H2- H2- H1- H1- identifier L1_H2- L2_H2- L1_H2- L2_H2- set L2 L2L1 L2 H1-L1 H1-L2 H2-L1 H2-L2 0 0 0 96.9 0 0.7 0 0.9 328*-327* 0 0 0 6.827.5 45.1 0 0 361*-313* 0 0 1 55.5 0 8.6 2 2.3 361*-340* 0 0 0 59.1 07.2 1.4 3  299-338* 0 0 2.6 54.6 4.6 11.2 0 0 368*-369* 0 0 0 43.6 1124.1 0 1.5 370*-371* 0 0 0 31.2 13.2 34.2 0 0 359*-360* 0 1.7 0 54.8 612.8 0 4.3 368*-372* 0 0 0 27.9 16.4 36.6 0 0 373*-374* 0 0 0 55.9 7.111.3 0 3.3 295-294 0 2.6 1.5 56.6 6.9 9.5 0 7.2 375*-376* 0 1.6 0 36.511.9 32.4 0 0 377*-378* 0 1.2 0 28.9 13.3 39.9 0 7.9 74-73 0 1.4 0 58.40 7 1.2 14.2 379*-380* 0 0 0 19.1 20.1 45.1 0 0 381*-382* 0 1.3 0 74.2 00 0 6 383*-384* 0 0 0 60.7 4.2 14.1 0 6.1 349*-338* 0 0 0 49.2 3.5 26.60 0 334*-333* 0 0 0 3.6 22.4 64.6 0 0 378*-377* 0 0 0 70.2 2.05 6.1 07.75 375*-385* 0 0 0 56 8.2 22.6 0 2.1 Unique identifier H1- H2- set Fabregion Design Type L1_Ab H1_mutation L1_mutation L2_Ab H2_mutation D3H44WT WT PERT WT 304*-306* constant combination D3H44 A139W_S186KF116A_Q124E_L135A_(—) PERT A139G_K145T_D146G_(—) (electrostatic + T180EQ179E_V190A steric) 362*-359* variable steric D3H44 W103F P44F PERT V37F386*-387* combination combination D3H44 Q39E_L124W Q38R_V133A PERTQ39R_L124A of constant (electrostatic + and variable steric) 388*-389*constant electrostatic D3H44 WT WT PERT K145L_Q179E 390*-391* constantelectrostatic D3H44 L143E_K145T_S188L Q124R PERT S186R 374*-392*variable steric D3H44 L45A P44W PERT WT 387*-386* combinationcombination D3H44 Q39R_L124A Q38E_V133F PERT Q39E_L124W of constant(electrostatic + and variable steric) 393*-394* constant electrostaticD3H44 K145T_S186E_S188L S131R PERT S186R 395*-396* constantelectrostatic D3H44 H172R WT PERT H172T Unique SEC step identifierH1:H2:L1:L2 performed Number of paired:mispaired paired:mispaired setL2_mutation DNA ratio post pA? experiments species(all) species (mean)WT 22:8:53:17 Y 2 3:97_2:98  2:98 304*-306* L135W 22:8:53:17 N 1 20:8020:80 362*-359* F98L 22:8:46:24 N 1 20:80 20:80 386*-387* Q38E_V133F22:8:46:24 N 1 19:81 19:81 388*-389* S131K 15:15:35:35 N 1 18:82 18:82390*-391* T178E_T180E 22:8:46:24 N 1 18:82 18:82 374*-392* WT 22:8:46:24N 1 18:82 18:82 387*-386* Q38R_V133A 22:8:46:24 N 1 18:82 18:82393*-394* Q160E_T178E 22:8:46:24 N 1 18:82 18:82 395*-396* S174R22:8:46:24 N 1 17:83 17:83 Δ (VAR- H1-L1_H2- Unique REF_WT) H1-L1_H2- L2(and H1- H1- H1- H1- H2- identifier paired_over_(—) paired_over_(—) L2(and H1- L2_H2-L1) L1_H1- L1_H1- L2_H1- L1_H2- set mispaired_Scalarmispaired_Scalar L2_H2-L1) side peak L1 L2 L2 L1 −3.72 0 1.55 notpresent 0 0 0 0 304*-306* −1.36 2.36 10.4 0.2 0 2.5 0 0 362*-359* −1.372.35 9 0.4 0 0 3 0 386*-387* −1.44 2.28 11.6 1.6 0 0 0 0 388*-389* −1.522.2 11.6 1.9 0 0 0 0 390*-391* −1.53 2.19 6.8 2.8 4.5 0 0 0 374*-392*−1.54 2.18 11.2 1.5 0 0 0 0 387*-386* −1.54 2.18 7.8 1.5 0 0 0 0393*-394* −1.54 2.18 7.1 1.1 5.3 0 1.2 0 395*-396* −1.61 2.11 4.6 1 1.50 3 0 Unique H2- H2- H1- H1- identifier L1_H2- L2_H2- L1_H2- L2_H2- setL2 L2 L1 L2 H1-L1 H1-L2 H2-L1 H2-L2 0 0 0 96.9 0 0.7 0 0.9 304*-306* 0 00 62.3 7.6 14.9 0 2.4 362*-359* 0 8.6 0 27 1.5 49.7 0 1.2 386*-387* 0 01.3 64.1 6.3 15.4 0 1.2 388*-389* 0 0 0 77.8 1.3 4.3 0 5 390*-391* 0 0 064.2 4 18 0 2.5 374*-392* 0 0 2.6 61.4 6.4 18.4 0 0 387*-386* 0 1.3 075.5 1.6 7 0 6.9 393*-394* 0 0 0 61.9 3.5 19.3 0 1.7 395*-396* 0 1.8 064.8 1.7 15.6 0 7 Unique identifier H1- H2- set Fab region Design TypeL1_Ab H1_mutation L1_mutation L2_Ab H2_mutation D3H44 WT WT PERT WT354*-353* constant combination D3H44 A139W_K145T_Q179E F116A_S131K PERTS186R (electrostatic + steric) 306*-304* constant combination *D3H44A139G_K145T_D146G_(—) L135W PERT A139W_S186K (electrostatic +Q179E_V190A steric) 397*-398* constant electrostatic D3H44K145T_S186E_S188L S131R PERT S186R 399*-400* constant electrostaticD3H44 K145T_S186E_S188L S131R PERT S186R 401*-402* constantelectrostatic D3H44 K145T_S186E_S188L S131R PERT S186R 403*-404*constant steric D3H44 A139W F116A PERT WT 382*-381* combinationcombination D3H44 Q39E Q38R_L135W PERT Q39R_A139W of constant(electrostatic + and variable steric) 405*-391* constant electrostaticD3H44 K145T_S186E_S188L Q124R PERT S186R 406*-407* constantelectrostatic D3H44 K145T_S186E_S188L S131R PERT S186R 408*-377*variable steric D3H44 WT WT PERT L45A 409*-410* constant steric D3H44L124W V133A PERT L124A 55-56 variable electrostatic vs D3H44 Q39M Q38MPERT Q39R hydrophobic 371*-370* combination combination D3H44Q39E_A139G_V190A Q38R_L135W PERT Q39R_A139W of constant (electrostatic +and variable steric) 377*-408* variable steric D3H44 L45A P44F PERT WT411*-384* constant electrostatic D3H44 H172R_T192K WT PERT H172T412*-413* constant electrostatic D3H44 K145T_S186E_S188L Q124R PERTS186R 53-54 variable electrostatic D3H44 Q39R Q38E PERT WT 414*-413*constant electrostatic D3H44 L143E_K145T_S188L Q124R PERT S186R415*-416* constant electrostatic D3H44 K145T_Q179E S131K PERT S186R417*-418* constant steric D3H44 A139W_V190I F116A_L135V PERT WT UniqueSEC step identifier H1:H2:L1:L2 performed Number of paired:mispairedpaired:mispaired set L2_mutation DNA ratio post pA? experimentsspecies(all) species (mean) WT 22:8:53:17 Y 2 3:97_2:98  2:98 354*-353*L135W_Q160E_T180E 22:8:46:24 N 1 16:84 16:84 306*-304*F116A_Q124E_L135A_(—) 22:8:53:17 N 1 16:84 16:84 T180E 397*-398* T178E22:8:46:24 N 1 15:85 15:85 399*-400* T178E_T180E 22:8:46:24 N 1 15:8515:85 401*-402* Q124E_T178E 22:8:46:24 N 1 14:86 14:86 403*-404* L135W22:8:46:24 N 1 13:87 13:87 382*-381* Q38E_F116A 22:8:46:24 N 1 13:8713:87 405*-391* T178E_T180E 22:8:46:24 N 1 13:87 13:87 406*-407*Q124E_Q160E_T180E 22:8:46:24 N 1 13:87 13:87 408*-377* P44F 22:8:46:24 N1 12:88 12:88 409*-410* V133F 22:8:46:24 N 1 11:89 11:89 55-56 Q38E22:8:46:24 N 1 11:89 11:89 371*-370* Q38E_F116A_L135V 22:8:46:24 N 111:89 11:89 377*-408* WT 22:8:46:24 N 1 11:89 11:89 411*-384*N137K_S174R 22:8:46:24 N 1 11:89 11:89 412*-413* Q160E_T178E 22:8:46:24N 1 11:89 11:89 53-54 WT 22:8:46:24 N 1 11:89 11:89 414*-413*Q160E_T178E 22:8:46:24 N 1 10:90 10:90 415*-416* Q160E_T180E 22:8:46:24N 1 10:90 10:90 417*-418* L135W 22:8:46:24 N 1 10:90 10:90 Δ (VAR-H1-L1_H2- Unique REF_WT) H1-L1_H2- L2 (and H1- H1- H1- H1- H2-identifier paired_over_(—) paired_over_(—) L2 (and H1- L2_H2-L1) L1_H1-L1_H1- L2_H1- L1_H2- set mispaired_Scalar mispaired_Scalar L2_H2-L1)side peak L1 L2 L2 L1 −3.72 0 1.55 not present 0 0 0 0 354*-353* −1.662.06 7.8 1.3 0 1.8 6.9 0 306*-304* −1.67 2.05 1.9 0.1 0 0 2.1 0397*-398* −1.72 2 7 1.3 4.8 0 1.3 0 399*-400* −1.76 1.96 8.4 1.5 0 0 0 0401*-402* −1.84 1.88 5.5 1 4.8 0 0 0 403*-404* −1.87 1.85 4.9 0.7 0 03.9 0 382*-381* −1.88 1.84 8.3 1.3 0 0 0 0 405*-391* −1.92 1.8 7.6 1.81.5 0 1.5 0 406*-407* −1.93 1.79 4 0.6 5.6 0 0 0 408*-377* −2.04 1.683.3 1.3 1 0 5.6 0 409*-410* −2.06 1.66 3.5 0.6 0 0 0 0 55-56 −2.1 1.624.9 1.9 0 0 0 0 371*-370* −2.11 1.61 6.7 0.7 0 0 0 0 377*-408* −2.111.61 4.8 0.6 0 0 2.7 0 411*-384* −2.12 1.6 5.4 0.7 0 6.5 0 0 412*-413*−2.13 1.59 5.5 1.4 0 0 2.4 0 53-54 −2.14 1.58 4.8 0.7 0 0 0 0 414*-413*−2.18 1.54 4.7 1.8 2.1 0 0 0 415*-416* −2.18 1.54 4.4 1 1.6 0 4.5 0417*-418* −2.19 1.53 5.3 0.4 0 0 3.2 0 Unique H2- H2- H1- H1- identifierL1_H2- L2_H2- L1_H2- L2_H2- set L2 L2 L1 L2 H1-L1 H1-L2 H2-L1 H2-L2 0 00 96.9 0 0.7 0 0.9 354*-353* 0 1 0 41.7 7.2 33.6 0 0 306*-304* 0 0 3.229.7 13.9 49.2 0 0 397*-398* 0 0 0 57.4 3.4 26.1 0 0 399*-400* 0 1.3 071.1 3.2 14 0 1.8 401*-402* 0 0 0 74.5 1.8 11.8 0 1.6 403*-404* 0 1.51.5 51.1 5.8 30.1 0 1.2 382*-381* 0 0 0 77.2 1.8 9.5 0 3.1 405*-391* 0 00 59.5 3.7 26.2 0 0 406*-407* 0 0 0 81.7 0 5.5 0 3.1 408*-377* 0 2.1 071.9 0 11 0 5.1 409*-410* 0 3.9 0 81.8 0 6.9 0 3.9 55-56 0 1.4 1.9 77.31.5 9.9 0 3.1 371*-370* 0 0 0 71.4 2.6 17.7 0 1.5 377*-408* 0 0 0 65.64.8 20.8 0 1.2 411*-384* 0 0 0 61.6 3.9 21.1 0 1.4 412*-413* 0 0 0 60.55.1 26.6 0 0 53-54 0 0 1.8 79.3 1.7 8.4 0 4 414*-413* 0 0 0 80.6 1.3 9.20 2.1 415*-416* 0 0 0 56.4 4.2 29 0 0 417*-418* 0 1.1 0 60.5 3.7 26.2 00 Unique identifier H1- H2- set Fab region Design Type L1_Ab H1_mutationL1_mutation L2_Ab H2_mutation D3H44 WT WT PERT WT 419*-420* constantelectrostatic D3H44 L143E_K145T_S188L Q124R PERT L143K 421*-422*constant electrostatic D3H44 WT WT PERT L143E_K145T 374*-373* variablesteric D3H44 L45A P44W PERT L45F 423*-418* constant steric D3H44 A139WF116A_L135V PERT WT 424*-260  constant electrostatic D3H44L143E_K145T_Q179E Q124K_T178R PERT S186R 425*-404* constant steric D3H44A139W_V190L F116A PERT WT 419*-426* constant electrostatic D3H44L143E_K145T_S188L Q124R PERT S186R 427*-418* constant steric D3H44A139W_V190L F116A_L135V PERT WT 428*-396* constant electrostatic D3H44H172R_T192K WT PERT H172T 392*-374* variable steric D3H44 WT WT PERTL45A 429*-430* constant electrostatic D3H44 K145T_S186E_S188L S131R PERTS186R 54-53 variable electrostatic D3H44 WT WT PERT Q39R 431*-418*constant steric D3H44 A139W_G141L_V190S F116A_L135V PERT WT 432*-426*constant electrostatic D3H44 K145T_S186E_S188L Q124R PERT S186R433*-434* constant steric D3H44 A139W_G141L_V190S F116A_F118L_L135V PERTWT 435*-283  constant electrostatic D3H44 L143E_K145T_S188L Q124R PERTS186R 56-55 variable electrostatic vs D3H44 Q39R Q38E PERT Q39Mhydrophobic 435*-436* constant electrostatic D3H44 L143E_K145T_S188LQ124R PERT L143K 437*-326* constant electrostatic D3H44 K145T_Q179ES131K PERT S186R 438*-285  constant electrostatic D3H44K145T_S186E_S188L Q124R PERT S186R 439*-283  constant electrostaticD3H44 K145T_S186E_S188L Q124R PERT S186R 440*-441* constantelectrostatic D3H44 K145T_S186E_S188L Q124R PERT S186R D3H44 WT WT TRASWT 331*-257  constant electrostatic D3H44 D146G_Q179K Q124E_Q160E_T180ETRAS L143E_K145T Unique SEC step identifier H1:H2:L1:L2 performed Numberof paired:mispaired paired:mispaired set L2_mutation DNA ratio post pA?experiments species(all) species (mean) WT 22:8:53:17 Y 2 3:97_2:98 2:98419*-420* Q124E_T178E 22:8:46:24 N 1 10:90  10:90  421*-422* Q124R15:15:35:35 N 1 10:90  10:90  374*-373* WT 22:8:46:24 N 1 9:91 9:91423*-418* L135W 22:8:46:24 N 1 9:91 9:91 424*-260  Q124E_Q160E_T180E22:8:46:24 N 1 9:91 9:91 425*-404* L135W 22:8:46:24 N 1 9:91 9:91419*-426* Q124E_T178E 22:8:46:24 N 1 9:91 9:91 427*-418* L135W22:8:46:24 N 1 9:91 9:91 428*-396* S174R 22:8:46:24 N 1 8:92 8:92392*-374* P44W 22:8:46:24 N 1 8:92 8:92 429*-430* Q160E_T180E 22:8:46:24N 1 8:92 8:92 54-53 Q38E 22:8:46:24 N 1 7:93 7:93 431*-418* L135W22:8:46:24 N 1 7:93 6:94 432*-426* Q124E_T178E 22:8:46:24 N 1 6:94 6:94433*-434* L135W 22:8:46:24 N 1 5:95 5:95 435*-283  T178E 22:8:46:24 N 15:95 5:95 56-55 Q38M 22:8:46:24 N 1 5:95 5:95 435*-436* T178E 22:8:46:24N 1 3:97 3:97 437*-326* Q124E_Q160E_T180E 22:8:46:24 N 1 3:97 3:97438*-285  Q124E_Q160E_T180E 22:8:46:24 N 1 2:98 2:98 439*-283  T178E22:8:46:24 N 1 2:98 2:98 440*-441* Q160E_T180E 22:8:46:24 N 1  0:100 0:100 WT 22:8:53:17 N 2 30:70_41:59 35:65  331*-257  Q124R_Q160K_T178R20:10:53:17 N 1 97:3  97:3  Δ (VAR- H1-L1_H2- Unique REF_WT) H1-L1_H2-L2 (and H1- H1- H1- H1- H2- identifier paired_over_(—) paired_over_(—)L2 (and H1- L2_H2-L1) L1_H1- L1_H1- L2_H1- L1_H2- set mispaired_Scalarmispaired_Scalar L2_H2-L1) side peak L1 L2 L2 L1 −3.72 0 1.55 notpresent 0 0 0 0 419*-420* −2.2 1.52 6.3 1.6 0 0 0 0 421*-422* −2.25 1.473.4 0.3 0 0 0 0 374*-373* −2.29 1.43 6.2 1.3 0 0 0 0 423*-418* −2.3 1.424 0.4 0 0 3.2 0 424*-260  −2.3 1.42 3.6 0.6 0 0 3.2 0 425*-404* −2.311.41 3.8 0.6 0 0 2.4 0 419*-426* −2.34 1.38 3.2 1 1.2 0 2.1 0 427*-418*−2.35 1.37 3.6 0.4 0 0 4.6 0 428*-396* −2.39 1.33 1.4 1.1 1.4 1.2 1.2 0392*-374* −2.44 1.28 3.3 1 0 0 0 0 429*-430* −2.45 1.27 2.5 0.3 4.2 01.3 0 54-53 −2.54 1.18 3 1 0 0 0 0 431*-418* −2.67 1.05 4.2 0.5 0 0 3.80 432*-426* −2.77 0.95 3.6 0.9 0 0 0 0 433*-434* −2.88 0.84 3.5 0.5 0 01.6 0 435*-283  −2.9 0.82 2.1 1 1.3 0 4.7 0 56-55 −3.03 0.69 1.6 0.6 0 01.2 0 435*-436* −3.41 0.31 1.4 0.5 0 0 2.1 0 437*-326* −3.58 0.14 0 0.41.5 0 3.1 0 438*-285  −3.75 −0.03 1.2 0.5 0 0 2.1 0 439*-283  −3.89−0.17 0 0.6 0 0 0 0 440*-441* −5 −1.28 0 0.4 0 0 1.1 0 −0.61 0 21.5 4.70 0 0 0 331*-257  3.62 4.23 63.3 1.7 2.5 0 0 0 Unique H2- H2- H1- H1-identifier L1_H2- L2_H2- L1_H2- L2_H2- set L2 L2 L1 L2 H1-L1 H1-L2 H2-L1H2-L2 0 0 0 96.9 0 0.7 0 0.9 419*-420* 0 0 0 78.2 2.5 11.8 0 1.2421*-422* 0 0 0 88.8 0 1.7 0 6.1 374*-373* 0 0 0 85.9 0 4.9 0 3423*-418* 0 1.9 0 61.5 2.1 26.2 0 1.1 424*-260  0 0 0 44.4 5.5 43.3 0 0425*-404* 0 2.6 0 75.9 1.6 12.6 0 1 419*-426* 0 0 0 58.6 3.3 30.5 0 1.1427*-418* 0 1.1 1.4 51.3 2.9 34 0 1.1 428*-396* 0 0 0 74.7 0 14.5 0 5.6392*-374* 0 0 0 86 0 6 0 4.7 429*-430* 0 0 0 65.1 1.2 25.6 0 0 54-53 0 00 72.6 4.3 20.1 0 0 431*-418* 0 0 0 66.7 2.3 23 0 0 432*-426* 0 0 0 75.22.3 18.9 0 0 433*-434* 0 0 1.2 80.6 1.8 11.3 0 0 435*-283  0 0 0 65.41.8 24.7 0 0 56-55 0 0 0 59.8 1.8 34.5 0 1.2 435*-436* 0 0 0 61.6 1.833.1 0 0 437*-326* 0 0 0 60 0 34.2 0 1.2 438*-285  0 0 0 58.9 1.1 36.8 00 439*-283  0 0 0 90.9 0 7.1 0 2 440*-441* 0 0 0 79.1 0 19.8 0 0 0 1.38.55 39.65 4.1 12.25 4.15 8.55 331*-257  0 0 2.6 0 31.6 0 0 0 Uniqueidentifier H1- H2- set Fab region Design Type L1_Ab H1_mutationL1_mutation L2_Ab H2_mutation D3H44 WT WT PERT WT 332*-284  constantelectrostatic D3H44 D146G_Q179K Q124E_Q160E_T180E TRAS L143E_K145T 90-92variable combination D3H44 V37W_Q39E Q38R_F98A TRAS V37I_Q39R(electrostatic + steric) 34-39 variable combination D3H44 V37W_Q39EQ38R_F98A TRAS Q39R (electrostatic + steric) 39-34 variable combinationD3H44 Q39R Q38E TRAS V37W_Q39E (electrostatic + steric) 442*-23 constant electrostatic D3H44 S115K_S156K_S186R Q124E_Q160E_T180E TRASK145L_Q179E 92-90 variable combination D3H44 V37I_Q39R Q38D_F98W TRASV37W_Q39E (electrostatic + steric)  284-332* constant electrostaticD3H44 L143E_K145T Q124R TRAS D146G_Q179K  257-331* constantelectrostatic D3H44 L143E_K145T Q124R_Q160K_T178R TRAS D146G_Q179K443*-326* constant electrostatic D3H44 S115K_K145L_S156K_Q179E S131KTRAS S186R D3H44 WT WT RAMU WT 332*-284  constant electrostatic D3H44D146G_Q179K Q124E_Q160E_T180E RAMU L143E_K145T 34-39 variablecombination D3H44 V37W_Q39E Q38R_F98A RAMU Q39R (electrostatic + steric) 284-332* constant electrostatic D3H44 L143E_K145T Q124R RAMUD146G_Q179K 90-92 variable combination D3H44 V37W_Q39E Q38R_F98A RAMUV37I_Q39R (electrostatic + steric) 442*-23  constant electrostatic D3H44S115K_S156K_S186R Q124E_Q160E_T180E RAMU K145L_Q179E  257-331* constantelectrostatic D3H44 L143E_K145T Q124R_Q160K_T178R RAMU D146G_Q179K331*-257  constant electrostatic D3H44 D146G_Q179K Q124E_Q160E_T180ERAMU L143E_K145T 443*-326* constant electrostatic D3H44S115K_K145L_S156K_Q179E S131K RAMU S186R Unique SEC step identifierH1:H2:L1:L2 performed Number of paired:mispaired paired:mispaired setL2_mutation DNA ratio post pA? experiments species(all) species (mean)WT 22:8:53:17 Y 2 3:97_2:98  2:98 332*-284  Q124R 20:10:53:17 N 1 88:1288:12 90-92 Q38D_F98W 20:10:53:17 N 1 86:14 86:14 34-39 Q38E 20:10:53:17N 1 81:19 81:19 39-34 Q38R_F98A 20:10:53:17 N 1 78:22 78:22 442*-23 S131K 20:10:53:17 N 1 72:28 72:28 92-90 Q38R_F98A 20:10:53:17 N 1 68:3268:32  284-332* Q124E_Q160E_T180E 20:10:53:17 N 1 58:42 57:43  257-331*Q124E_Q160E_T180E 20:10:53:17 N 1 47:53 47:53 443*-326*Q124E_Q160E_T180E 20:10:53:17 N 1 42:58 42:58 WT 22:8:53:17 N 225:75_42:58 33:67 332*-284  Q124R 20:10:53:17 N 1 82:18 82:18 34-39 Q38E20:10:53:17 N 1 82:18 82:18  284-332* Q124E_Q160E_T180E 20:10:53:17 N 175:25 75:25 90-92 Q38D_F98W 20:10:53:17 N 1 74:26 74:26 442*-23  S131K20:10:53:17 N 1 70:30 70:30  257-331* Q124E_Q160E_T180E 20:10:53:17 N 166:34 65:35 331*-257  Q124R_Q160K_T178R 20:10:53:17 N 1 58:42 58:42443*-326* Q124E_Q160E_T180E 20:10:53:17 N 1 41:59 41:59 Δ (VAR-H1-L1_H2- Unique REF_WT) H1-L1_H2- L2 (and H1- H1- H1- H1- H2-identifier paired_over_(—) paired_over_(—) L2 (and H1- L2_H2-L1) L1_H1-L1_H1- L2_H1- L1_H2- set mispaired_Scalar mispaired_Scalar L2_H2-L1)side peak L1 L2 L2 L1 −3.72 0 1.55 not present 0 0 0 0 332*-284  1.982.59 48.2 1.6 2.1 0 0 0 90-92 1.84 2.45 52.4 4.6 0 0 0 0 34-39 1.42 2.0356.9 4.3 0 0 0 0 39-34 1.25 1.86 42 5.4 0 0 0 0 442*-23  0.92 1.53 37.74 0 0 0 0 92-90 0.75 1.36 56.4 9 0 0 0 0  284-332* 0.3 0.91 17.2 1.8 0 00 0  257-331* −0.11 0.5 8.9 0.8 0 0 0 0 443*-326* −0.34 0.27 33.8 2.5 00 0 0 −0.695 −0.01 24.85 3.9 0 0.95 0.95 0 332*-284  1.54 2.24 55.3 81.9 3.7 0 0 34-39 1.49 2.19 61 8 0 0 0 0  284-332* 1.08 1.78 45.3 7.9 00 0 0 90-92 1.04 1.74 49 6.6 1.1 0 0 0 442*-23  0.87 1.57 41.2 9.2 0 0 00  257-331* 0.64 1.34 22.1 3.9 0 0 0 0 331*-257  0.32 1.01 26.1 3.7 3.32.3 0 0 443*-326* −0.37 0.32 29.2 3.9 0 0 0 0 Unique H2- H2- H1- H1-identifier L1_H2- L2_H2- L1_H2- L2_H2- set L2 L2 L1 L2 H1-L1 H1-L2 H2-L1H2-L2 0 0 0 96.9 0 0.7 0 0.9 332*-284  0 0 1.9 4.6 37.6 5.6 0 0 90-923.7 2.1 3.9 0 4.8 2.7 3.4 26.9 34-39 1.3 0 3 10.5 3.5 3.3 1.3 20.2 39-340 2.6 5.3 10.5 4 1.7 4.7 29.1 442*-23  0 1.2 13.7 0 4.3 0 14.8 28.392-90 1 0 11.2 15.4 7.3 3.1 1.5 4.2  284-332* 1.1 8.3 0 34.1 2.4 5.8 1.429.6  257-331* 0 5 0 45.3 0 7.4 0 33.4 443*-326* 0 0 1.6 46.1 7.8 10.7 00 0 0 0 27.25 1.6 30.5 6.55 7.35 332*-284  0 0 12.4 0 19.9 1.6 0 5.234-39 0 0 5 9.1 8 2.5 1.8 12.5  284-332* 0 0 4 14.7 3.8 1.7 5 25.5 90-920 0 17.6 1.2 9.4 0 7.4 14.3 442*-23  0 0 14.7 0 2 0 14.8 27.2  257-331*0 0 1 30.4 2.2 1.7 1.4 41.2 331*-257  0 0 36.3 0 25 0 3.4 3.6 443*-326*0 0 13.1 33.8 8.7 11 1.3 2.9 Unique identifier H1- H2- set Fab regionDesign Type L1_Ab H1_mutation L1_mutation L2_Ab H2_mutation D3H44 WT WTPERT WT 39-34 variable combination D3H44 Q39R Q38E RAMU V37W_Q39E(electrostatic + steric) 92-90 variable combination D3H44 V37I_Q39RQ38D_F98W RAMU V37W_Q39E (electrostatic + steric) TRAS WT WT RAMU WT 257-331* constant electrostatic TRAS L143E_K145T Q124R_Q160K_T178R RAMUD146G_Q179K  23-326* constant electrostatic TRAS K145L_Q179E S131K RAMUS186R  284-332* constant electrostatic TRAS L143E_K145T Q124R RAMUD146G_Q179K 34-39 variable combination TRAS V37W_Q39E Q38R_F98A RAMUQ39R (electrostatic + steric) 326*-23  constant electrostatic TRAS S186RQ124E_Q160E_T180E RAMU K145L_Q179E 92-90 variable combination TRASV37I_Q39R Q38D_F98W RAMU V37W_Q39E (electrostatic + steric) 90-92variable combination TRAS V37W_Q39E Q38R_F98A RAMU V37I_Q39R(electrostatic + steric) 332*-284  constant electrostatic TRASD146G_Q179K Q124E_Q160E_T180E RAMU L143E_K145T 39-34 variablecombination TRAS Q39R Q38E RAMU V37W_Q39E (electrostatic + steric)331*-257  constant electrostatic TRAS D146G_Q179K Q124E_Q160E_T180E RAMUL143E_K145T Unique SEC step identifier H1:H2:L1:L2 performed Number ofpaired:mispaired paired:mispaired set L2_mutation DNA ratio post pA?experiments species(all) species (mean) WT 22:8:53:17 Y 2 3:97_2:98 2:98 39-34 Q38R_F98A 20:10:53:17 N 1 38:62 38:62 92-90 Q38R_F98A20:10:53:17 N 1 23:77 22:78 WT 8:22:35:35 N 2 46:54_52:48 49:51 257-331* Q124E_Q160E_T180E 8:22:35:35 N 1 93:7  93:7   23-326*Q124E_Q160E_T180E 8:22:35:35 N 1 89:11 89:11  284-332* Q124E_Q160E_T180E8:22:35:35 N 1 79:21 79:21 34-39 Q38E 8:22:35:35 N 1 42:58 42:58326*-23  S131K 8:22:35:35 N 1 35:65 35:65 92-90 Q38R_F98A 8:22:35:35 N 131:69 31:69 90-92 Q38D_F98W 8:22:35:35 N 1 26:74 26:74 332*-284  Q124R8:22:35:35 N 1 22:78 22:78 39-34 Q38R_F98A 8:22:35:35 N 1 13:87 13:87331*-257  Q124R_Q160K_T178R 8:22:35:35 N 1  5:95  5:95 Δ (VAR- H1-L1_H2-Unique REF_WT) H1-L1_H2- L2 (and H1- H1- H1- H1- H2- identifierpaired_over_(—) paired_over_(—) L2 (and H1- L2_H2-L1) L1_H1- L1_H1-L2_H1- L1_H2- set mispaired_Scalar mispaired_Scalar L2_H2-L1) side peakL1 L2 L2 L1 −3.72 0 1.55 not present 0 0 0 0 39-34 −0.48 0.21 19 3.8 5.13.9 0 0 92-90 −1.24 −0.55 8 1.6 4 1.7 0 0 −0.05 0 40.8 6.3 0 0 0 0 257-331* 2.51 2.56 63.1 6.9 0 0 0 0  23-326* 2.09 2.14 58.4 2.1 0 0 0 0 284-332* 1.34 1.39 45.5 4.5 0 0 0 0 34-39 −0.33 −0.28 31.5 3.6 0 0 0 0326*-23  −0.64 −0.59 23.3 1 0 0 0 0 92-90 −0.79 −0.74 20.3 1.5 0 0 0 090-92 −1.04 −0.99 14.4 1.4 0 0 0 0 332*-284  −1.29 −1.24 12.3 1.2 0 0 00 39-34 −1.94 −1.89 7.9 1.1 0 0 0 1.1 331*-257  −2.9 −2.85 2.7 0.4 0 0 01.1 Unique H2- H2- H1- H1- identifier L1_H2- L2_H2- L1_H2- L2_H2- set L2L2 L1 L2 H1-L1 H1-L2 H2-L1 H2-L2 0 0 0 96.9 0 0.7 0 0.9 39-34 0 0 35.5 05.5 1.6 20.7 8.7 92-90 0 0 58.2 0 7.2 0 17.6 3.3 0 0 15.8 13.9 1.55 3.1518.4 6.45  257-331* 0 0 4.2 1.2 2.1 0 2.1 27.4  23-326* 0 1.6 5.2 2.2 00 3.6 29  284-332* 0 0 11 0 1.3 0 9.8 32.3 34-39 0 0 46.2 0 2.1 0 12 8.3326*-23  0 0 45.7 0 1.2 0 19.8 10.1 92-90 0 0 45.2 0 1.6 0 23.6 9.290-92 0 0 57.5 1.1 5.2 0 15.3 6.4 332*-284  0 0 53.1 0 2.2 0 25.3 7.139-34 0 0 62.1 0 1.5 0 24.1 3.2 331*-257  0 0 51.6 0 0 0 42.2 2.5

TABLE 23 Unique identifier Fab Design H1- set region Type L1_AbH1_mutation L1_mutation 340*-339* variable steric D3H44 V37W_W103F F98A340*-339* variable steric D3H44 V37W_W103F F98A 340*-339* variablesteric D3H44 V37W_W103F F98A 340*-339* variable steric D3H44 V37W_W103FF98A 340*-339* variable steric D3H44 V37W_W103F F98A 340*-339* variablesteric D3H44 V37W_W103F F98A 340*-339* variable steric D3H44 V37W_W103FF98A 340*-339* variable steric D3H44 V37W_W103F F98A 340*-339* variablesteric D3H44 V37W_W103F F98A SEC step Unique performed identifierH1:H2:L1:L2 post set H2-L2_Ab H2_mutation L2_mutation DNA ratio pA?340*-339* PERT W103V P44W 18:12:35:35 N 340*-339* PERT W103V P44W18:12:46:24 N 340*-339* PERT W103V P44W 18:12:56:14 N 340*-339* PERTW103V P44W 22:8:35:35 N 340*-339* PERT W103V P44W 22:8:46:24 N 340*-339*PERT W103V P44W 22:8:56:14 N 340*-339* PERT W103V P44W 26:4:35:35 N340*-339* PERT W103V P44W 26:4:46:24 N 340*-339* PERT W103V P44W26:4:56:14 N H1-L1_H2-L2 Unique H1-L1_H2- (and H1- H1- identifier Numberof paired:mispaired paired:mispaired L2 (and H1- L2_H2-L1) L1_H1- setexperiments species (all) species (mean) paired_over_mispaired_ScalarL2_H2-L1) side peak L1 340*-339* 1 84:16 84:16 1.66 77.2 5.7 0 340*-339*1 87:13 87:13 1.87 80.5 6.4 0 340*-339* 1 86:14 86:14 1.84 36.8 3.4 2.1340*-339* 1 78:22 78:22 1.25 33 2.8 2 340*-339* 2 80:20_81:19 81:191.425 31.35 3.7 0.9 340*-339* 1 89:11 89:11 2.07 34.5 3.2 0 340*-339* 175:25 75:25 1.1 4.7 0.8 7.5 340*-339* 1 81:19 81:19 1.47 7.9 1 6.9340*-339* 1 92:8  92:8  2.47 12.2 1.6 3.2 Unique H1- H1- H2- H2- H2- H1-H1- identifier L1_H1- L2_H1- L1_H2- L1_H2- L2_H2- L1_H2- L2_H2- set L2L2 L1 L2 L2 L1 L2 H1-L1 H1-L2 H2-L1 H2-L2 340*-339* 0 0 0 1.7 2 2.6 11.71.1 0 0 3.6 340*-339* 0 0 0 1.9 3.4 7.1 4.4 0 0 0 2.8 340*-339* 0 0 01.4 1.8 6 1.1 45.5 5.2 0 0 340*-339* 0 0 0 0 1.3 1.1 6.6 41.5 14.6 0 0340*-339* 0 0 0 1.9 2.95 5 2.9 44.65 8.8 0.75 0.8 340*-339* 0 0 0 1.52.8 4.7 0 51.3 5 0 0 340*-339* 2.9 0 0 0 0 0 1.1 62.7 21 0 0 340*-339*1.8 0 0 0 0 0 1.3 66.5 15.6 0 0 340*-339* 0 0 0 0 1.9 2 0 74.9 5.8 0 0

TABLE 24 Unique identifier Fab Design H1- set region Type L1_AbH1_mutation L1_mutation 335*-336* combination combination D3H44V37W_L124E F98A_V133A_S176K of (electrostatic + constant steric) andvariable 333*-334* combination combination D3H44 Q39E_S186RQ38R_Q160E_T180E of (electrostatic) constant and variable 326*-23 constant electrostatic D3H44 S186R Q124E_Q160E_T180E 331*-257  constantelectrostatic D3H44 D146G_Q179K Q124E_Q160E_T180E 154-152 variablecombination D3H44 V37W_Q39E Q38R_F98A (electrostatic + steric) 313*-337*variable steric D3H44 V37W F98A 340*-337* variable steric D3H44V37W_W103F F98A 336*-335* combination combination D3H44 L124RF98W_V133G_S176D of (electrostatic + constant steric) and variable 92-90variable combination *D3H44 V37I_Q39R Q38D_F98W (electrostatic + steric)66-67 constant electrostatic D3H44 L143A_D146G_Q179RQ124E_V133W_Q160E_T180E 340*-339* variable steric D3H44 V37W_W103F F98A329*-330* constant combination *D3H44 A139G_V190A L135W (electrostatic +steric) 329*-330* constant combination *D3H44 A139G_V190A L135W(electrostatic + steric)  300-349* variable steric D3H44 V37W F98A152-154 variable combination D3H44 V37A_Q39R_W103V Q38E_P44W(electrostatic + steric) 92-90 variable combination *D3H44 V37I_Q39RQ38D_F98W (electrostatic + steric) 34-39 variable combination D3H44V37W_Q39E Q38R_F98A (electrostatic + steric) 332*-284  constantelectrostatic D3H44 D146G_Q179K Q124E_Q160E_T180E 331*-257  constantelectrostatic D3H44 D146G_Q179K Q124E_Q160E_T180E 90-92 variablecombination D3H44 V37W_Q39E Q38R_F98A (electrostatic + steric) 92-90variable combination D3H44 V37I_Q39R Q38D_F98W (electrostatic + steric)34-39 variable combination D3H44 V37W_Q39E Q38R_F98A (electrostatic +steric) 39-34 variable combination D3H44 Q39R Q38E (electrostatic +steric) 442*-23  constant electrostatic D3H44 S115K_S156K_S186RQ124E_Q160E_T180E  257-331* constant electrostatic D3H44 L143E_K145TQ124R_Q160K_T178R  284-332* constant electrostatic D3H44 L143E_K145TQ124R 443*-326* constant electrostatic D3H44 S115K_K145L_S156K_Q179ES131K 34-39 variable combination D3H44 V37W_Q39E Q38R_F98A(electrostatic + steric) 332*-284  constant electrostatic D3H44D146G_Q179K Q124E_Q160E_T180E  257-331* constant electrostatic D3H44L143E_K145T Q124R_Q160K_T178R  284-332* constant electrostatic D3H44L143E_K145T Q124R 90-92 variable combination D3H44 V37W_Q39E Q38R_F98A(electrostatic + steric) 442*-23  constant electrostatic D3H44S115K_S156K_S186R Q124E_Q160E_T180E 331*-257  constant electrostaticD3H44 D146G_Q179K Q124E_Q160E_T180E 443*-326* constant electrostaticD3H44 S115K_K145L_S156K_Q179E S131K 92-90 variable combination D3H44V37I_Q39R Q38D_F98W (electrostatic + steric) 39-34 variable combinationD3H44 Q39R Q38E (electrostatic + steric)  257-331* constantelectrostatic TRAS L143E_K145T Q124R_Q160K_T178R  23-326* constantelectrostatic TRAS K145L_Q179E S131K  284-332* constant electrostaticTRAS L143E_K145T Q124R 92-90 variable combination TRAS V37I_Q39RQ38D_F98W (electrostatic + steric) 34-39 variable combination TRASV37W_Q39E Q38R_F98A (electrostatic + steric) 90-92 variable combinationTRAS V37W_Q39E Q38R_F98A (electrostatic + steric) 332*-284  constantelectrostatic TRAS D146G_Q179K Q124E_Q160E_T180E 326*-23  constantelectrostatic TRAS S186R Q124E_Q160E_T180E 39-34 variable combinationTRAS Q39R Q38E (electrostatic + steric) 331*-257  constant electrostaticTRAS D146G_Q179K Q124E_Q160E_T180E Unique identifier H2- H1:H2:L1:L2 setL2_Ab H2_mutation L2_mutation DNA ratio 335*-336* PERT L124RF98W_V133G_S176D 22:8:35:35 333*-334* PERT Q39R_K145T_Q179E Q38E_S131K22:8:46:24 326*-23  PERT K145L_Q179E S131K 22:8:56:14 331*-257  PERTL143E_K145T Q124R_Q160K_T178R 22:8:46:24 154-152 PERT V37A_Q39R_W103VQ38E_P44W 15:15:35:35 313*-337* PERT L45A P44W 22:8:46:24 340*-337* PERTL45A P44W 15:15:35:35 336*-335* PERT V37W_L124E F98A_V133A_S176K22:8:46:24 92-90 PERT V37W_Q39E Q38R_F98A 22:8:35:35 66-67 PERTK145T_Q179D_S188F V133A_Q160K_T178R 18:12:35:35 340*-339* PERT W103VP44W 18:12:46:24 329*-330* PERT A139W_K145Y_Q179E F116A_S131K_L135A20:10:46:24 329*-330* PERT A139W_K145Y_Q179E F116A_S131K_L135A22:8:46:24  300-349* PERT W103F P44F 22:8:46:24 152-154 PERT V37W_Q39EQ38R_F98A 26:4:56:14 92-90 PERT V37W_Q39E Q38R_F98A 20:10:40:30 34-39PERT Q39R Q38E 22:8:46:24 332*-284  TRAS L143E_K145T Q124R 20:10:53:17331*-257  TRAS L143E_K145T Q124R_Q160K_T178R 20:10:53:17 90-92 TRASV37I_Q39R Q38D_F98W 20:10:53:17 92-90 TRAS V37W_Q39E Q38R_F98A20:10:53:17 34-39 TRAS Q39R Q38E 20:10:53:17 39-34 TRAS V37W_Q39EQ38R_F98A 20:10:53:17 442*-23  TRAS K145L_Q179E S131K 20:10:53:17 257-331* TRAS D146G_Q179K Q124E_Q160E_T180E 20:10:53:17  284-332* TRASD146G_Q179K Q124E_Q160E_T180E 20:10:53:17 443*-326* TRAS S186RQ124E_Q160E_T180E 20:10:53:17 34-39 RAMU Q39R Q38E 20:10:53:17 332*-284 RAMU L143E_K145T Q124R 20:10:53:17  257-331* RAMU D146G_Q179KQ124E_Q160E_T180E 20:10:53:17  284-332* RAMU D146G_Q179KQ124E_Q160E_T180E 20:10:53:17 90-92 RAMU V37I_Q39R Q38D_F98W 20:10:53:17442*-23  RAMU K145L_Q179E S131K 20:10:53:17 331*-257  RAMU L143E_K145TQ124R_Q160K_T178R 20:10:53:17 443*-326* RAMU S186R Q124E_Q160E_T180E20:10:53:17 92-90 RAMU V37W_Q39E Q38R_F98A 20:10:53:17 39-34 RAMUV37W_Q39E Q38R_F98A 20:10:53:17  257-331* RAMU D146G_Q179KQ124E_Q160E_T180E 8:22:35:35  23-326* RAMU S186R Q124E_Q160E_T180E8:22:35:35  284-332* RAMU D146G_Q179K Q124E_Q160E_T180E 8:22:35:35 92-90RAMU V37W_Q39E Q38R_F98A 8:22:35:35 34-39 RAMU Q39R Q38E 8:22:35:3590-92 RAMU V37I_Q39R Q38D_F98W 8:22:35:35 332*-284  RAMU L143E_K145TQ124R 8:22:35:35 326*-23  RAMU K145L_Q179E S131K 8:22:35:35 39-34 RAMUV37W_Q39E Q38R_F98A 8:22:35:35 331*-257  RAMU L143E_K145TQ124R_Q160K_T178R 8:22:35:35 Unique SEC step identifier performed Numberof paired:mispaired set post pA? experiments speciespaired_over_mispaired_Scalar 335*-336* N 1 100:0 5 333*-334* N 1 100:0 5326*-23  N 1 99:1 4.41 331*-257  N 1 99:1 4.41 154-152 N 1 98:2 3.89313*-337* N 1 97:3 3.62 340*-337* N 1 96:4 3.08 336*-335* N 1 93:7 2.5792-90 N 1 92:8 2.42 66-67 N 1 91:9 2.27 340*-339* N 1 85:15 1.72329*-330* N 1 83:17 1.56 329*-330* N 1 83:17 1.56  300-349* N 1 77:231.2 152-154 N 1 71:29 0.91 92-90 N 1 68:32 0.75 34-39 N 1 19:81 −1.46332*-284  N 1 96:4  3.23 331*-257  N 1 88:12 1.96 90-92 N 1 87:13 1.8792-90 N 1 81:19 1.45 34-39 N 1 79:21 1.31 39-34 N 1 74:26 1.06 442*-23 N 1 68:32 0.75  257-331* N 1 64:36 0.58  284-332* N 1 49:51 −0.04443*-326* N 1 44:56 −0.24 34-39 N 1 81:19 1.42 332*-284  N 1 80:20 1.41 257-331* N 1 80:20 1.39  284-332* N 1 77:23 1.23 90-92 N 1 74:26 1.03442*-23  N 1 69:31 0.78 331*-257  N 1 58:42 0.32 443*-326* N 1 41:59−0.37 92-90 N 1 39:61 −0.45 39-34 N 1 34:66 −0.68  257-331* N 1 96:4 3.03  23-326* N 1 91:9  2.29  284-332* N 1 79:21 1.33 92-90 N 1 38:62−0.51 34-39 N 1 37:63 −0.55 90-92 N 1 20:80 −1.4 332*-284  N 1 13:87−1.89 326*-23  N 1 11:89 −2.12 39-34 N 1  7:93 −2.6 331*-257  N 1  2:98−3.75 H1-L1_H2-L2 Unique H1-L1_H2-L2 (and H1- H1- H1- H1- identifier(and H1- L2_H2-L1) L1_H1- L1_H1- L2_H1- set L2_H2-L1) side peak L1 L2 L2335*-336* 88.6 12 0 0 0 333*-334* 73.2 5 0 0 0 326*-23  12 0.6 5.9 0 0331*-257  2.7 0.3 10.3 0 0 154-152 67.3 2.6 0 0 0 313*-337* 57.5 3.6 1 00 340*-337* 68.3 5.6 0 0 0 336*-335* 71.1 7.2 0 0 0 92-90 42.5 3 1.4 0 066-67 22.8 1 3.3 0 0 340*-339* 55.5 4.5 0 0 0 329*-330* 26.3 1.3 0 0 0329*-330* 19.5 1.1 1.1 0 0  300-349* 34.8 1.9 0 0 0 152-154 47.2 2.5 0 00 92-90 41.3 1 0 0 0 34-39 2.2 0.9 0 0 0 332*-284  47.4 1.4 2 0 0331*-257  61.7 2.2 0 0 0 90-92 66.8 4.7 0 0 0 92-90 32.1 4.7 0 0 0 34-3959.7 5.5 0 0 0 39-34 56.6 8.1 0 0 0 442*-23  41.4 3.8 1.2 1.7 0 257-331* 31.4 2.8 0 0 0  284-332* 28.4 3.5 0 0 0 443*-326* 31.3 2.3 0 00 34-39 61.2 7.6 0 0 0 332*-284  58.9 7.6 2.1 4.9 0  257-331* 12.7 2 0 00  284-332* 19.5 3.5 0 0 0 90-92 40.5 5.1 0 0 0 442*-23  28.2 6 0 0 0331*-257  29.5 4 3.4 2.5 0 443*-326* 22.3 3 0 0 0 92-90 22.2 4.1 4.7 2.70 39-34 10.2 1.7 2.7 1.7 0  257-331* 54.6 5.2 0 0 0  23-326* 28.4 1.3 00 0  284-332* 57.9 5.5 0 0 0 92-90 14.6 1.3 0 0 0 34-39 17.8 1.8 0 0 090-92 7.2 0.8 0 0 0 332*-284  5.6 0.7 0 0 0 326*-23  5.1 0.3 0 0 0 39-342.7 0.6 0 0 0 331*-257  1.1 0.2 0 0 0 Unique H2- H2- H2- H1- H1-identifier L1_H2- L1_H2- L2_H2- L1_H2- L2_H2- set L1 L2 L2 L1 L2 H1-L1H1-L2 H2-L1 H2-L2 335*-336* 0 0 1.1 0 0 7.1 0 0 3.3 333*-334* 0 0 0 0 024.3 0 0 2.4 326*-23  0 0 0 0 0 80.8 1.2 0 0 331*-257  0 0 0 0 0 85.91.2 0 0 154-152 0 0 0 0 0 25.9 2 0 4.8 313*-337* 0 0 0 0 0 37.7 2.6 01.1 340*-337* 0 0 0 0 4.4 1.2 0 0 26.1 336*-335* 0 0 4.4 1.8 4.2 4.1 01.1 13.2 Unique SEC step identifier performed Number of paired:mispairedset post pA? experiments species paired_over_mispaired_Scalar 92-90 01.5 0 1.2 66-67 0 0 0 1 340*-339* 0 0 1.2 7.3 329*-330* 0 0 1.6 1.5329*-330* 0 0 0 0  300-349* 0 0 1.2 0 152-154 0 0 0 12.3 92-90 0 0 0 034-39 0 0 0 77.2 332*-284  0 0 0 0 331*-257  0 0 0 12.4 90-92 0 4.8 03.3 92-90 0 0 4.5 2.6 34-39 0 2.7 0 1.7 39-34 0 0 0 9.9 442*-23  0 0 016  257-331* 0 2.8 4.5 0  284-332* 0 2.5 9.3 0 443*-326* 0 0 0 1.3 34-390 0 0 5 332*-284  0 0 0 13.1  257-331* 0 0 1.6 0  284-332* 0 0 2.8 1.990-92 0 0 0 10.4 442*-23  0 0 0 10.6 331*-257  0 0 0 35.3 443*-326* 0 00 6.9 92-90 0 0 0 42.9 39-34 1.1 0 0 21  257-331* 0 0 0 2.4  23-326* 0 03.3 2.1  284-332* 0 0 0 15.6 92-90 0 0 0 22.9 34-39 0 0 0 31.8 90-92 0 00 45 332*-284  1.1 0 0 34.4 326*-23  1.6 0 0 37.1 39-34 0 0 0 81.4331*-257  1.1 0 0 55.2 H1-L1_H2-L2 Unique H1-L1_H2-L2 (and H1- H1- H1-H1- identifier (and H1- L2_H2-L1) L1_H1- L1_H1- L2_H1- set L2_H2-L1)side peak L1 L2 L2 92-90 1.9 46.2 3.6 0 1.9 66-67 0 64.5 8.4 0 0340*-339* 2.3 1.9 0 5.6 26.2 329*-330* 11.9 1.9 0 3.9 52.8 329*-330* 6.260.3 11.2 0 1.8  300-349* 23.1 0 0 0 40.9 152-154 10.2 24.1 6.1 0 092-90 20.2 23.3 12 0 3.3 34-39 0 16.6 0 4 0 332*-284  1.4 46.7 2.4 0 0331*-257  0 25.9 0 0 0 90-92 1.2 7.4 2.5 1.5 12.5 92-90 12.4 0 0 4 44.334-39 13.1 4.3 3.7 0 14.7 39-34 11 4.5 1.8 3.1 13.1 442*-23  0 3.2 014.3 22.1  257-331* 26.8 1.8 6.4 0 26.3  284-332* 43.3 6 5.1 0 5.4443*-326* 40.1 12.8 14.4 0 0 34-39 9.2 5.6 2.3 2.9 13.8 332*-284  0 13.91.6 0 5.5  257-331* 18.7 1.2 0 1.3 64.5  284-332* 8.8 0 0 11.9 55 90-921.7 2.5 0 14.2 30.8 442*-23  0 1.3 0 20.7 39.1 331*-257  0 20.7 0 4.34.2 443*-326* 23.8 18.7 28.4 0 0 92-90 0 5.7 1.9 13.6 6.4 39-34 0 3.31.8 40.8 17.5  257-331* 0 1.7 0 2.2 39.2  23-326* 0 0 0 7.1 59.1 284-332* 0 2.4 0 5.4 18.8 92-90 1.2 0 0 38.3 23 34-39 0 1.2 0 31.7 17.590-92 0 1.8 0 35.2 10.8 332*-284  0 0 0 51.4 7.5 326*-23  0 0 0 50.5 5.639-34 0 2.9 0 11.7 1.3 331*-257  0 0 0 41.4 1.2 Unique H2- H2- H2- H1-H1- identifier L1_H2- L1_H2- L2_H2- L1_H2- L2_H2- set L1 L2 L2 L1 L2H1-L1 H1-L2 H2-L1 H2-L2 335*-336* 0 0 1.1 0 0 7.1 0 0 3.3 333*-334* 0 00 0 0 24.3 0 0 2.4 326*-23  0 0 0 0 0 80.8 1.2 0 0 331*-257  0 0 0 0 085.9 1.2 0 0 154-152 0 0 0 0 0 25.9 2 0 4.8 313*-337* 0 0 0 0 0 37.7 2.60 1.1 340*-337* 0 0 0 0 4.4 1.2 0 0 26.1 336*-335* 0 0 4.4 1.8 4.2 4.1 01.1 13.2 92-90 0 1.5 0 1.2 1.9 46.2 3.6 0 1.9 66-67 0 0 0 1 0 64.5 8.4 00 340*-339* 0 0 1.2 7.3 2.3 1.9 0 5.6 26.2 329*-330* 0 0 1.6 1.5 11.91.9 0 3.9 52.8 329*-330* 0 0 0 0 6.2 60.3 11.2 0 1.8  300-349* 0 0 1.2 023.1 0 0 0 40.9 152-154 0 0 0 12.3 10.2 24.1 6.1 0 0 92-90 0 0 0 0 20.223.3 12 0 3.3 34-39 0 0 0 77.2 0 16.6 0 4 0 332*-284  0 0 0 0 1.4 46.72.4 0 0 331*-257  0 0 0 12.4 0 25.9 0 0 0 90-92 0 4.8 0 3.3 1.2 7.4 2.51.5 12.5 92-90 0 0 4.5 2.6 12.4 0 0 4 44.3 34-39 0 2.7 0 1.7 13.1 4.33.7 0 14.7 39-34 0 0 0 9.9 11 4.5 1.8 3.1 13.1 442*-23  0 0 0 16 0 3.2 014.3 22.1  257-331* 0 2.8 4.5 0 26.8 1.8 6.4 0 26.3  284-332* 0 2.5 9.30 43.3 6 5.1 0 5.4 443*-326* 0 0 0 1.3 40.1 12.8 14.4 0 0 34-39 0 0 0 59.2 5.6 2.3 2.9 13.8 332*-284  0 0 0 13.1 0 13.9 1.6 0 5.5  257-331* 0 01.6 0 18.7 1.2 0 1.3 64.5  284-332* 0 0 2.8 1.9 8.8 0 0 11.9 55 90-92 00 0 10.4 1.7 2.5 0 14.2 30.8 442*-23  0 0 0 10.6 0 1.3 0 20.7 39.1331*-257  0 0 0 35.3 0 20.7 0 4.3 4.2 443*-326* 0 0 0 6.9 23.8 18.7 28.40 0 92-90 0 0 0 42.9 0 5.7 1.9 13.6 6.4 39-34 1.1 0 0 21 0 3.3 1.8 40.817.5  257-331* 0 0 0 2.4 0 1.7 0 2.2 39.2  23-326* 0 0 3.3 2.1 0 0 0 7.159.1  284-332* 0 0 0 15.6 0 2.4 0 5.4 18.8 92-90 0 0 0 22.9 1.2 0 0 38.323 34-39 0 0 0 31.8 0 1.2 0 31.7 17.5 90-92 0 0 0 45 0 1.8 0 35.2 10.8332*-284  1.1 0 0 34.4 0 0 0 51.4 7.5 326*-23  1.6 0 0 37.1 0 0 0 50.55.6 39-34 0 0 0 81.4 0 2.9 0 11.7 1.3 331*-257  1.1 0 0 55.2 0 0 0 41.41.2

TABLE 25 SEC puri- Antigen Antigen Unique H1- fication affin- affin-identifier L1_H2-L2 performed ity −TF, ity −HER2, set Tm (° C.) pH postpA? KD (nM) KD(nM)  300-349* 69.66, 79.94 7 y ND ND 340*-337*   69,75.54 7 y 1.31 2.8 340*-339*   69, 75.94 7 y 1.34 12.9  326*-23  77.16 7y 0.03 4.14 92-90 70.42, 73   7 y 0.01 7.09 313*-337* 70.65, 78.26 7 y0.9 3.29 333*-334* 72.07 7 y 0.02 1.02 335*-336* 69.5, 76.5 7 y 1.044.71 336*-335*  65.2, 75.35 7 y 0.01 16.7  329*-330* 72.57 7 n 0.04 3.69# 329*-330*  71.99 7 n 0.04 5.16 # 92-90  70.53 7 n 0.03 10.4  154-15275.5  7 n 0.6 NB  300-349* 70.62, 80.1  7 n 0.4 6.49 333*-334* 72.89 4 nND ND 327*-328* 74.69 4 n ND ND 313*-339* 77.53 4 n ND ND 338*-299 75.98 4 n ND ND 313*-337* 77.39 4 n ND ND 331*-257  77.83 4 n ND ND332*-284  77.22 4 n ND ND 326*-23  78.53 4 n ND ND 92-90 69.6, 72  4 nND ND 34-39 73.35 4 n ND ND 325*-31  78.17 4 n ND ND 305*-307* 78.35 4 nND ND 90-92 70.43 4 n ND ND

TABLE 26 H1-L1_Ab H1_mutation L1_mutation L1_tag H2-L2_Ab H2_mutationD3H44 WT WT HA PERT WT D3H44 WT WT none PERT WT D3H44 WT WT FLAG PERT WTSEC step performed H1:H2:L1:L2 post Number of H1-L1_Ab L2_mutationL2_tag DNA ratio pA? experiments D3H44 WT FLAG 22:8:53:17 Y 2 D3H44 WTFLAG 22:8:53:17 Y 1 D3H44 WT HA 22:8:53:17 Y 1 H1-L1_H2- H1-L1_H2-L2 L2(and H1- H1- H1- paired:mispaired Pair'ed:mispaired (and H1- L2_H2-L1)L1_H1- L1_H1- H1-L1_Ab species (all) species (mean)paired_over_mispaired_Scalar L2_H2-L1) side peak L1 L2 D3H44 3:97_2:982:98 −3.72 1.55 0 0 D3H44 18:82 18:82  −1.5 18.2 0 0 D3H44  1:99 1:99−4.59 0 0 1.3 H1- H2- H2- H2- H1- H1- L2_H1- L1_H2- L1_H2- L2_H2- L1_H2-L2_H2- H1-L1_Ab L2 L1 L2 L2 L1 L2 H1-L1 H1-L2 H2-L1 H2-L2 D3H44 0 0 0 00 96.9 0 0.7 0 0.9 D3H44 0 0 0 0 5.4 76.4 0 0 0 0 D3H44 0 0 10 1 2 85.60 0 0 0

TABLE 27 Unique identifier set H1_mutation L1_mutation H2_mutationL2_mutation 92-90 V37I_Q39R Q38D_F98W V37W_Q39E Q38R_F98A 111-112Q39D_A139G_V190A Q38R_L135W Q39R_A139W Q38D_F116A_L135A 57-58L143K_D146G Q124E_V133D L143E_K145T Q124R 66-67 L143A_D146G_Q179RQ124E_V133W_Q160E_T180E K145T_Q179D_S188F V133A_Q160K_T178R 106-97 V37I_Q39D Q38R_F98W V37W_Q39R_W103F Q38E_F98L 1-2 S186RQ124E_Q160E_T178D K145L_Q179E S131K 152-154 V37A_Q39R_W103V Q38E_P44WV37W_Q39E Q38R_F98A 72-65 D146G_Q179R Q124E_Q160E_T178DK145T_Q179D_S188L Q160K_T178R 107-108 A139G_V190A L135W A139WF116A_L135A

TABLE 30 Unique identifier set H1_mutation L1_mutation H2_mutationL2_mutation 164-165 A139G_K145L_Q179E_V190A S131R_L135W A139WF116A_L135A 236-237 A139G_K145L_Q179E_V190A S131R_L135W A139WF116S_L135A  306*-304*, A139G_K145T_D146G_Q179E_(—) L135W A139W_S186KF116A_Q124E_L135A_(—) 304*-306* V190A T180E 208-209 A139G_S188G_V190AL135W_S176L_T178S A139W_S188H_V190S F116S_L135A_S176A 215-216A139G_S188G_V190A V133G_L135W_S176F_(—) A139W_S188A F116S_L135A_S176AT178A 194-195 A139G_V190A F98W_L135W V37W_W103H_A139W F98L_F116A_L135A 107-108, A139G_V190A L135W A139W F116A_L135A 108-107 A139G_V190A L135WA139W F116A_L135V 329*-330* A139G_V190A L135W A139W_K145Y_Q179EF116A_S131K_L135A 103-104 A139G_V190A L135W_N137A A139W F116A_L135A211-212 A139I F118W_V133S A139S L135R 224-225 A139I F118W_V133SA139V_V190S WT 139-140 A139I_K145T_D146G_Q179E_(—) F116A_V133G_S176F_(—)S186K_S188H_V190G F118W_Q124E_V133S_(—) S188G_V190S T178AS176A_T178S_T180E 231-232 A139I_K145T_D146G_S188G_(—)F116A_V133G_S176F_(—) S186K_S188H_V190G F118W_V133S_S176A_(—) V190ST178A T178S_T180E A139I_V190S F116A A139V F118W_V133S 246-225A139I_V190S WT A139I F118W_V133S A139V F118W_V133S A139W_V190S F116AA139V F118W_V133S V190G F116A 314*-315* A139V_K145L_Q179E_S188G_(—)F116A_S131K_V133G_(—) A139W_S186K_S188A F118W_V133S_S176A_T180E V190SS176F_T178A A139V_V190S F116A A139V F118W_V133S 403*-404* A139W F116A WTL135W 423*-418* A139W F116A_L135V WT L135W A139W F118W_V133S V190G F116S 346*-345*, A139W F98W_F116A V37W F98A_L135W 345*-346* 196-197 A139WF98W_F116A_L135A V37W_W103H_A139G_(—) F98L_L135W V190A 433*-434*A139W_G141L_V190S F116A_F118L_L135V WT L135W 431*-418* A139W_G141L_V190SF116A_L135V WT L135W  354*-353*, A139W_K145T_Q179E F116A_S131K S186RL135W_Q160E_T180E 353*-354* A139W_V190A F116A A139W WT A139W_V190A F116AWT WT 417*-418* A139W_V190I F116A_L135V WT L135W 425*-404* A139W_V190LF116A WT L135W 427*-418* A139W_V190L F116A_L135V WT L135W A139W_V190SF116A A139W WT A139W_V190S F116A WT WT A139W_V190S F116S A139WF118W_V133S 64-78 D146G_Q179K Q124E_Q160E_T178D K145E_D146G_Q179D_(—)Q160K_T178R S188L 69-78 D146G_Q179K Q124E_Q160E_T178D K145T_Q179D_S188FQ160K_T178R 65-78 D146G_Q179K Q124E_Q160E_T178D K145T_Q179D_S188LQ160K_T178R 61-71 D146G_Q179K Q124E_Q160E_T178D L143E_K145TQ124R_Q160K_T178R 113-78  D146G_Q179K Q124E_Q160E_T178D L143E_K145TQ160K_T178R 5--6 D146G_Q179K Q124E_Q160E_T180E K145E_D146G_Q179D_(—)Q160K_T178R S188L 23-24 D146G_Q179K Q124E_Q160E_T180E K145L_Q179E S131K9--5 D146G_Q179K Q124E_Q160E_T180E K145T_Q179D_S188L Q160K_T178R332*-284,  D146G_Q179K Q124E_Q160E_T180E L143E_K145T Q124R  284-332*331*-257,  D146G_Q179K Q124E_Q160E_T180E L143E_K145T Q124R_Q160K_T178R 257-331*  5-59 D146G_Q179K Q124E_Q160E_T180E L143E_K145T Q160K_T178R201-83  D146G_Q179K Q124E_V133W_Q160E_(—) K145E_D146G_Q179D_(—)Q160K_T178R T180E S188F 80-83 D146G_Q179K Q124E_V133W_Q160E_(—)K145T_Q179D_S188F Q160K_T178R T180E 85-83 D146G_Q179KQ124E_V133W_Q160E_(—) K145T_Q179D_S188L Q160K_T178R T180E 72-64D146G_Q179R Q124E_Q160E_T178D K145E_D146G_Q179D_(—) Q160K_T178R S188L234-238 D146G_Q179R Q124E_Q160E_T178D K145E_D146G_Q179D_(—) T178R S188L72-69 D146G_Q179R Q124E_Q160E_T178D K145T_Q179D_S188F Q160K_T178R 72-65,65-72 D146G_Q179R Q124E_Q160E_T178D K145T_Q179D_S188L Q160K_T178R 60-61D146G_Q179R Q124E_Q160E_T178D L143E_K145T Q124R_Q160K_T178R  72-113D146G_Q179R Q124E_Q160E_T178D L143E_K145T Q160K_T178R 7--6 D146G_Q179RQ124E_Q160E_T180E K145E_D146G_Q179D_(—) Q160K_T178R S188L 7--9D146G_Q179R Q124E_Q160E_T180E K145T_Q179D_S188L Q160K_T178R  7-59D146G_Q179R Q124E_Q160E_T180E L143E_K145T Q160K_T178R  81-201D146G_Q179R Q124E_V133W_Q160E_(—) K145E_D146G_Q179D_(—) Q160K_T178RT180E S188F  79-114 D146G_Q179R Q124E_V133W_Q160E_(—)K145E_D146G_Q179D_(—) V133A_Q160K_T178R T180E S188F 80-81 D146G_Q179RQ124E_V133W_Q160E_(—) K145T_Q179D_S188F Q160K_T178R T180E 67-79D146G_Q179R Q124E_V133W_Q160E_(—) K145T_Q179D_S188F V133A_Q160K_T178RT180E 85-81 D146G_Q179R Q124E_V133W_Q160E_(—) K145T_Q179D_S188LQ160K_T178R T180E 68-79 D146G_Q179R Q124E_V133W_Q160E_(—)K145T_Q179D_S188L V133A_Q160K_T178R T180E 63-64 D146G_S186RQ124E_Q160E_T178D K145E_D146G_Q179D_(—) Q160K_T178R S188L 234-235D146G_S186R Q124E_Q160E_T178D K145E_D146G_Q179D_(—) T178R S188L 63-69D146G_S186R Q124E_Q160E_T178D K145T_Q179D_S188F Q160K_T178R 63-65D146G_S186R Q124E_Q160E_T178D K145T_Q179D_S188L Q160K_T178R 62-61D146G_S186R Q124E_Q160E_T178D L143E_K145T Q124R_Q160K_T178R  63-113D146G_S186R Q124E_Q160E_T178D L143E_K145T Q160K_T178R 8--6 D146G_S186RQ124E_Q160E_T180E K145E_D146G_Q179D_(—) Q160K_T178R S188L 8--9D146G_S186R Q124E_Q160E_T180E K145T_Q179D_S188L Q160K_T178R  8-59D146G_S186R Q124E_Q160E_T180E L143E_K145T Q160K_T178R 207-203F100M_W103H P44W_L89W V37W_F100W F98A 207-205 F100M_W103H P44W_L89WV37W_F100W_W103L F98A 202-203 F100M_W103V P44W_L89W V37W_F100W F98A202-205 F100M_W103V P44W_L89W V37W_F100W_W103L F98A F100W F98L V37I WTF100W F98L WT WT 296-297 F100W F98M W103F Y36W 344*-121,  F100W_W103FF98L Q39R Q38E_F98W  121-344* 355*-186,  F100W_W103F F98L WT F98W 186-355* 298-297 F100W_W103F F98M W103F Y36W 254-255 F174A_S188G S176WF174G_S188A WT 10--11 F174V_P175S_S188G S176L F174V_S188L S176G  3-70F174V_P175S_S188G S176L F174V_S188L V133S 12--13 F174V_P175S_S188G S176LF174V_S188L WT 18--11 F174V_P175S_S188G S176L F174W_S188L S176G  3-133F174V_P175S_S188G S176L F174W_S188L V133S 12-15 F174V_P175S_S188G S176LF174W_S188L WT 20-11 F174V_P175S_S188G S176L S188L S176G 3-4F174V_P175S_S188G S176L S188L V133S 12-14 F174V_P175S_S188G S176L S188LWT 3-19, 19--3, F174V_P175S_S188G S176L S188L_V190Y V133S 19-3 383*-384* H172R WT H172T N137K_S174R 395*-396* H172R WT H172T S174R411*-384* H172R_T192K WT H172T N137K_S174R 428*-396* H172R_T192K WTH172T S174R  82-201 K145E_D146G_Q179D_S188F Q160K_T178RL143A_D146G_Q179R Q124E_V133W_Q160E_T180E  66-114K145E_D146G_Q179D_S188F V133A_Q160K_T178R L143A_D146G_Q179RQ124E_V133W_Q160E_T180E 1--2, K145L_Q179E S131K S186R Q124E_Q160E_T178D1-2   23-326*, K145L_Q179E S131K S186R Q124E_Q160E_T180E 326*-23, 442*-23,  443*-326* 388*-389* K145L_Q179E S131K WT WT 80-82K145T_Q179D_S188F Q160K_T178R L143A_D146G_Q179R Q124E_V133W_Q160E_T180E66-67 K145T_Q179D_S188F V133A_Q160K_T178R L143A_D146G_Q179RQ124E_V133W_Q160E_T180E 85-82 K145T_Q179D_S188L Q160K_T178RL143A_D146G_Q179R Q124E_V133W_Q160E_T180E 66-68 K145T_Q179D_S188LV133A_Q160K_T178R L143A_D146G_Q179R Q124E_V133W_Q160E_T180E 437*-326*K145T_Q179E S131K S186R Q124E_Q160E_T180E 415*-416* K145T_Q179E S131KS186R Q160E_T180E 438*-285  K145T_S186E_S188L Q124R S186RQ124E_Q160E_T180E 432*-426* K145T_S186E_S188L Q124R S186R Q124E_T178E412*-413* K145T_S186E_S188L Q124R S186R Q160E_T178E 440*-441*K145T_S186E_S188L Q124R S186R Q160E_T180E 439*-283  K145T_S186E_S188LQ124R S186R T178E 405*-391* K145T_S186E_S188L Q124R S186R T178E_T180E406*-407* K145T_S186E_S188L S131R S186R Q124E_Q160E_T180E 401*-402*K145T_S186E_S188L S131R S186R Q124E_T178E 393*-394* K145T_S186E_S188LS131R S186R Q160E_T178E 429*-430* K145T_S186E_S188L S131R S186RQ160E_T180E 397*-398* K145T_S186E_S188L S131R S186R T178E 399*-400*K145T_S186E_S188L S131R S186R T178E_T180E 409*-410* L124A V133F L124WV133A  294-295, L124E V133A_S176K L124R V133G_S176D 295-294 288-289L124E V133G_S176R L124R V133A_S176D 263-264 L124E V133G_S176R L124RV133G_S176D 271-272 L124E_H172R V133A_S176K L124R_H172AV133A_S174W_S176D 267-268 L124E_H172R V133G_S176R L124R_H172AV133A_S174W_S176D 261-262 L124E_H172R V133G_S176R L124R_H172AV133G_S174W_S176D 275-276 L124E_H172W V133A_S176K L124R_H172TV133G_S174R_S176D 286-287 L124E_H172W V133G_S176R L124R_H172TV133A_S174R_S176D 269-270 L124E_H172W V133G_S176R L124R_H172TV133G_S174R_S176D  336*-335*, L124R F98W_V133G_S176D V37W_L124EF98A_V133A_S176K 335*-336* 308*-320* L124S WT L124W F118A 265-266L143E_K145T Q124K_T178R S186R Q124E 259-260 L143E_K145T Q124K_T178RS186R Q124E_Q160E_T180E 277-281 L143E_K145T Q124R L143K Q124E 58-57,57-58 L143E_K145T Q124R L143K_D146G Q124E_V133D 277-278 L143E_K145TQ124R L143R Q124E 277-280 L143E_K145T Q124R S186R Q124E 366*-367*L143E_K145T Q124R S186R Q124E_Q160E_T178D 284-285 L143E_K145T Q124RS186R Q124E_Q160E_T180E 282-283 L143E_K145T Q124R S186R T178E 277-279L143E_K145T Q124R WT Q124E 421*-422* L143E_K145T Q124R WT WT 257-258L143E_K145T Q124R_Q160K_T178R S186R Q124E_Q160E_T180E 424*-260 L143E_K145T_Q179E Q124K_T178R S186R Q124E_Q160E_T180E 379*-380*L143E_K145T_Q179E Q124K_T178R S186R Q124E_T178E_T180E 290-291L143E_K145T_S188L Q124K L143K Q124E 290-293 L143E_K145T_S188L Q124KL143R Q124E 290-292 L143E_K145T_S188L Q124K S186R Q124E 303-281L143E_K145T_S188L Q124R L143K Q124E 419*-420* L143E_K145T_S188L Q124RL143K Q124E_T178E 375*-385* L143E_K145T_S188L Q124R L143K Q124E_V133E435*-436* L143E_K145T_S188L Q124R L143K T178E 368*-369*L143E_K145T_S188L Q124R L143K V133E 303-278 L143E_K145T_S188L Q124RL143R Q124E 375*-376* L143E_K145T_S188L Q124R L143R Q124E_V133E368*-372* L143E_K145T_S188L Q124R L143R V133E 303-280 L143E_K145T_S188LQ124R S186R Q124E 419*-426* L143E_K145T_S188L Q124R S186R Q124E_T178E414*-413* L143E_K145T_S188L Q124R S186R Q160E_T178E 435*-283 L143E_K145T_S188L Q124R S186R T178E 390*-391* L143E_K145T_S188L Q124RS186R T178E_T180E 303-279 L143E_K145T_S188L Q124R WT Q124E  377*-378*,L45A P44F L45F WT 378*-377*  358*-359*, L45A P44F V37F F98L 359*-358* 299-300, L45A P44F V37W F98A 300-299  299-338*, L45A P44F V37W_W103FF98A 338*-299   377*-408*, L45A P44F WT WT 408*-377*  374*-373*, L45AP44W L45F WT 373*-374*  364*-356*, L45A P44W V37F F98L 356*-364* 337*-313*, L45A P44W V37W F98A 313*-337*  337*-340*, L45A P44WV37W_W103F F98A 340*-337*  374*-392*, L45A P44W WT WT 392*-374* 249-250L45W Y87G V37A_W103H P44W  86-184 Q39D Q38N_T85K Q39K Q38N_T85E 21-22Q39D Q38R Q39R Q38D 75-76 Q39D Q38R Q39R Q38D_F98W 16-84 Q39D Q38R Q39RQ38E_F98W 161-142 Q39D Q38R V37A_Q39R_W103V Q38D_P44W 162-163 Q39D Q38RV37A_Q39R_W103V Q38E_P44W 75-77 Q39D Q38R V37I_Q39R Q38D_F98W 16-17 Q39DQ38R V37I_Q39R Q38E_F98W 135-136 Q39D Q38R V37W_Q39R Q38D_F98A 35-36Q39D Q38R V37W_Q39R Q38E_F98A 137-138 Q39D Q38R_F98W V37W_Q39R Q38D_F98A41-42 Q39D Q38R_F98W V37W_Q39R Q38E_F98A 145-147 Q39D Q38R_F98WV37W_Q39R_W103F Q38D_F98L 96-97 Q39D Q38R_F98W V37W_Q39R_W103F Q38E_F98L145-146 Q39D Q38R_F98W V37W_Q39R_W103H Q38D_F98L  96-132 Q39D Q38R_F98WV37W_Q39R_W103H Q38E_F98L 111-112 Q39D_A139G_V190A Q38R_L135W Q39R_A139WQ38D_F116A_L135A 192-193 Q39D_A139W Q38R_F116A_L135A Q39R_A139G_V190AQ38D_L135W 86-87 Q39E Q38N_T85K Q39K Q38N_T85E 127-128 Q39E Q38N_T85KV37W_Q39K Q38N_T85E_F98A 233-131 Q39E Q38N_T85K_F98W V37W_Q39KQ38N_T85E_F98A 239-243 Q39E Q38N_T85K_F98W V37W_Q39K_W103FQ38N_T85E_F98L 239-240 Q39E Q38N_T85K_F98W V37W_Q39K_W103HQ38N_T85E_F98L 301-302 Q39E Q38N_T85R Q39R Q38E_T85E 22-44 Q39E Q38RQ39R Q38D 99-76 Q39E Q38R Q39R Q38D_F98W 52-51, 51-52 Q39E Q38R Q39RQ38E 43-84 Q39E Q38R Q39R Q38E_F98W 141-142 Q39E Q38R V37A_Q39R_W103VQ38D_P44W 168-163 Q39E Q38R V37A_Q39R_W103V Q38E_P44W 99-77 Q39E Q38RV37I_Q39R Q38D_F98W 43-17 Q39E Q38R V37I_Q39R Q38E_F98W 190-191 Q39EQ38R V37W F98A 206-136 Q39E Q38R V37W_Q39R Q38D_F98A 37-36 Q39E Q38RV37W_Q39R Q38E_F98A 273-274 Q39E Q38R_F98W Q39R_F100W_W103F Q38E_F98M210-138 Q39E Q38R_F98W V37W_Q39R Q38D_F98A 105-42  Q39E Q38R_F98WV37W_Q39R Q38E_F98A 200-147 Q39E Q38R_F98W V37W_Q39R_W103F Q38D_F98L126-97  Q39E Q38R_F98W V37W_Q39R_W103F Q38E_F98L 200-146 Q39E Q38R_F98WV37W_Q39R_W103H Q38D_F98L 126-132 Q39E Q38R_F98W V37W_Q39R_W103HQ38E_F98L  382*-381*, Q39E Q38R_L135W Q39R_A139W Q38E_F116A 381*-382* 371*-370*, Q39E_A139G_V190A Q38R_L135W Q39R_A139W Q38E_F116A_L135V370*-371*  386*-387*, Q39E_L124W Q38R_V133A Q39R_L124A Q38E_V133F387*-386*  327*-328*, Q39E_S186R Q38R_Q124E_Q160E_(—) Q39R_K145L_Q179EQ38E_S131K 328*-327* T180E  333*-334*, Q39E_S186R Q38R_Q160E_T180EQ39R_K145T_Q179E Q38E_S131K 334*-333* 170-171 Q39K Q38N_T85EV37A_Q39E_W103H Q38N_P44W_T85K 170-172 Q39K Q38N_T85E V37A_Q39E_W103VQ38N_P44W_T85K 86-88 Q39K Q38N_T85E V37I_Q39E Q38N_T85K 119-120 Q39KQ38N_T85E_F98W V37W_Q39E Q38N_T85K_F98A 226-228 Q39K Q38N_T85E_F98WV37W_Q39E_W103F Q38N_T85K_F98L 226-227 Q39K Q38N_T85E_F98WV37W_Q39E_W103H Q38N_T85K_F98L 55-56, 56-55 Q39M Q38M Q39R Q38E  22-151Q39R Q38D V37I_Q39R Q38R 45-28 Q39R Q38D V37W_Q39D Q38R_F98A 27-28 Q39RQ38D V37W_Q39E Q38R_F98A 89-98 Q39R Q38D_F98W V37W_Q39D Q38R_F98A115-122 Q39R Q38D_F98W V37W_Q39D_W103F Q38R_F98L 115-217 Q39R Q38D_F98WV37W_Q39D_W103H Q38R_F98L 89-90 Q39R Q38D_F98W V37W_Q39E Q38R_F98A115-116 Q39R Q38D_F98W V37W_Q39E_W103F Q38R_F98L 115-218 Q39R Q38D_F98WV37W_Q39E_W103H Q38R_F98L 123-124 Q39R Q38D_F98W V37W_W103F F98L 123-219Q39R Q38D_F98W V37W_W103H F98L  307*-305*, Q39R Q38E V37W F98A 305*-307*39-40 Q39R Q38E V37W_Q39D Q38R_F98A 39-34, 34-39 Q39R Q38E V37W_Q39EQ38R_F98A 53-54, 54-53 Q39R Q38E WT WT 93-38 Q39R Q38E_F98W V37W_Q39DQ38R_F98A 118-91  Q39R Q38E_F98W V37W_Q39D_W103F Q38R_F98L 118-143 Q39RQ38E_F98W V37W_Q39D_W103H Q38R_F98L 93-26, 26-93 Q39R Q38E_F98WV37W_Q39E Q38R_F98A 118-74  Q39R Q38E_F98W V37W_Q39E_W103F Q38R_F98L118-144 Q39R Q38E_F98W V37W_Q39E_W103H Q38R_F98L 121-95  Q39R Q38E_F98WV37W_W103F F98L 121-175 Q39R Q38E_F98W V37W_W103H F98L  342*-341*, S186RF98W_Q160E_T180E V37W_K145T_Q179E F98A_S131K 341*-342* S188I WT WTS176V_T178L 198-199 V37A_Q39E_W103H Q38N_P44W_T85K V37W_Q39KQ38N_T85E_F98A 220-199 V37A_Q39E_W103V Q38N_P44W_T85K V37W_Q39KQ38N_T85E_F98A 213-214 V37A_Q39K_W103H Q38N_P44W_T85E V37W_Q39EQ38N_T85K_F98A 173-174 V37A_Q39R_W103V Q38D_P44W V37W F98A 148-149V37A_Q39R_W103V Q38D_P44W V37W_Q39D Q38R_F98A 148-150 V37A_Q39R_W103VQ38D_P44W V37W_Q39E Q38R_F98A 158-159 V37A_Q39R_W103V Q38E_P44W V37WF98A 152-153 V37A_Q39R_W103V Q38E_P44W V37W_Q39D Q38R_F98A  152-154,V37A_Q39R_W103V Q38E_P44W V37W_Q39E Q38R_F98A 154-152 V37A_W103H P44WV37A_W103V F98L V37A_W103H P44W V37A_W103V F98W V37A_W103H P44WV37I_F100W F98L 242-244 V37A_W103H P44W V37T_A93Q_W103T F98L 250-256V37A_W103H P44W V37T_A93Q_W103T Y87G V37A_W103H P44W V37W F98A 241-242V37A_W103H P44W V37W_W103H F98L 177-157 V37A_W103H P44W_F98W V37W F98A221-222 V37A_W103H_A139G_V190A P44W_L135W V37W_A139W F98A_F116A_L135AV37A_W103V P44W V37W F98A 189-157 V37A_W103V P44W_F98W V37W F98A 101-102V37E L89R_F98T WT WT 229-230 V37E L89R_F98V V37S-A93K F98Y 109-110 V37EL89R_F98V WT WT  182-183, V37E_F100D L89R_F98W V37S_A93K F98Y  182-317*47-48, 48-47, V37E_F100D L89R_F98W WT WT  319*-310*, 309*-310* 359*-362*, V37F F98L W103F P44F 362*-359*  356*-357*, V37F F98L W103FP44W 357*-356*  359*-360*, V37F F98L W103V P44F 360*-359* 356*-365* V37FF98L W103V P44W 352*-186,  V37F F98L WT F98W  186-352* 363*-186, V37F_W103F F98L WT F98W  186-363* V37I F98W V37W F98A V37I F98WV37W_W103F F98L 187-188 V37I F98W V37W_W103H F98L V37I WT V37W F98A155-36  V37I_Q39D Q38R V37W_Q39R Q38E_F98A 185-138 V37I_Q39D Q38R_F98WV37W_Q39R Q38D_F98A 50-42 V37I_Q39D Q38R_F98W V37W_Q39R Q38E_F98A106-97  V37I_Q39D Q38R_F98W V37W_Q39R_W103F Q38E_F98L 106-132 V37I_Q39DQ38R_F98W V37W_Q39R_W103H Q38E_F98L 129-128 V37I_Q39E Q38N_T85KV37W_Q39K Q38N_T85E_F98A 130-131 V37I_Q39E Q38N_T85K_F98W V37W_Q39KQ38N_T85E_F98A 251-243 V37I_Q39E Q38N_T85K_F98W V37W_Q39K_W103FQ38N_T85E_F98L 251-240 V37I_Q39E Q38N_T85K_F98W V37W_Q39K_W103HQ38N_T85E_F98L 134-36  V37I_Q39E Q38R V37W_Q39R Q38E_F98A 181-138V37I_Q39E Q38R_F98W V37W_Q39R Q38D_F98A 49-42 V37I_Q39E Q38R_F98WV37W_Q39R Q38E_F98A 160-147 V37I_Q39E Q38R_F98W V37W_Q39R_W103FQ38D_F98L 100-97  V37I_Q39E Q38R_F98W V37W_Q39R_W103F Q38E_F98L 160-146V37I_Q39E Q38R_F98W V37W_Q39R_W103H Q38D_F98L 100-132 V37I_Q39EQ38R_F98W V37W_Q39R_W103H Q38E_F98L 253-120 V37I_Q39K Q38N_T85E_F98WV37W_Q39E Q38N_T85K_F98A 252-228 V37I_Q39K Q38N_T85E_F98WV37W_Q39E_W103F Q38N_T85K_F98L 252-227 V37I_Q39K Q38N_T85E_F98WV37W_Q39E_W103H Q38N_T85K_F98L 92-98 V37I_Q39R Q38D_F98W V37W_Q39DQ38R_F98A 117-122 V37I_Q39R Q38D_F98W V37W_Q39D_W103F Q38R_F98L 117-217V37I_Q39R Q38D_F98W V37W_Q39D_W103H Q38R_F98L 92-90, 90-92 V37I_Q39RQ38D_F98W V37W_Q39E Q38R_F98A 117-116 V37I_Q39R Q38D_F98WV37W_Q39E_W103F Q38R_F98L 117-218 V37I_Q39R Q38D_F98W V37W_Q39E_W103HQ38R_F98L 125-124 V37I_Q39R Q38D_F98W V37W_W103F F98L 125-219 V37I_Q39RQ38D_F98W V37W_W103H F98L 33-40 V37I_Q39R Q38E V37W_Q39D Q38R_F98A 33-34V37I_Q39R Q38E V37W_Q39E Q38R_F98A 25-38 V37I_Q39R Q38E_F98W V37W_Q39DQ38R_F98A 73-91 V37I_Q39R Q38E_F98W V37W_Q39D_W103F Q38R_F98L  73-143V37I_Q39R Q38E_F98W V37W_Q39D_W103H Q38R_F98L 25-26 V37I_Q39R Q38E_F98WV37W_Q39E Q38R_F98A 73-74, 74-73 V37I_Q39R Q38E_F98W V37W_Q39E_W103FQ38R_F98L  73-144 V37I_Q39R Q38E_F98W V37W_Q39E_W103H Q38R_F98L 94-95V37I_Q39R Q38E_F98W V37W_W103F F98L  94-175 V37I_Q39R Q38E_F98WV37W_W103H F98L 166-157 V37I_W103H P44W_F98W V37W F98A 156-157V37T_A93Q_W103L P44W_F98W V37W F98A 169-157 V37T_A93Q_W103T P44W_F98WV37W F98A 223-179 V37T_A93Q_W103T P44W_L89W_F98A V37W_F100W F98A 167-157V37T_A93Q_W103V P44W_F98W V37W F98A 204-179 V37T_A93Q_W103VP44W_L89W_F98A V37W_F100W F98A  300-349*, V37W F98A W103F P44F 349*-300  313*-361*, V37W F98A W103F P44W 361*-313* 157-176 V37W F98A W103HP44W_F98W  300-343*, V37W F98A W103V P44F 343*-300   313*-339*, V37WF98A W103V P44W 339*-313* 157-178 V37W F98A W103V P44W_F98W 325*-31, V37W F98A WT F98W  31-325* 46-30 V37W F98A WT L89F_F98W  351*-312*, V37WF98A WT WT 312*-351* 31-32 V37W_A93V F98A WT F98W 29-30 V37W_A93V F98AWT L89F_F98W 179-245 V37W_F100W F98A W103H P44W_L89W_F98A  179-180,V37W_F100W F98A W103V P44W_L89W_F98A 323*-324* 247-248 V37W_F100W_W103LL89W_F98A V37W_F100W F98A  347*-348*, V37W_K145T_Q179E F98A_S131K WTF98W 348*-347*  338*-349*, V37W_W103F F98A W103F P44F 349*-338* 340*-361*, V37W_W103F F98A W103F P44W 361*-340*  338*-343*, V37W_W103FF98A W103V P44F 343*-338*  340*-339*, V37W_W103F F98A W103V P44W339*-340* 350*-31,  V37W_W103F F98A WT F98W  31-350* V37W_W103F F98L WTF98W 186-187 V37W_W103H F98L WT F98W

TABLE 31 H1_mutaton L1_mutation H2_mutation L2_mutation A139W F116A WTL135W H172T S174R H172R WT K145T_Q179E S131K and/or S186R and/or T178Eand/or and/or T178R and/or Q179K and/or Q124E and/or L143E_K145T Q124RL143K T180E or and/or Q160E and/or K145T_S186E V133E F174V_P175S_(—)S176L S188L V133S S188G S188G S176L S188L S176A L124A V133F L124W V133AL124E V133G_S176R L124R V133A_S176D V37W F98A WT WT F100W F98L WT WTQ39E Q38R Q39R Q38E Q39E Q38N_T85K Q39K Q38N_T85E Q39M Q38M Q39R Q38EF100W F98M W103F Y36W V37E L89R_F98T WT WT L45A P44F WT WT W103V P44WV37W F98A

Key for Tables 14-27 and 30-31 Table 14. LCCA data for D3H44 system, inFab pair format Table 15. Additional LCCA data for D3H44 system, in Fabpair format Table 16. LCCA data for selected pure and mixed systemsTable 17. LCCA data for selected pure and mixed systems, in Fab pairformat Table 18. WT LCCA_data for selected pure and mixed systems Table19. Table 17 data summary Table 20. Table 18 data summary Table 21. Mabassay data for D3H44 system Table 22. SMCA data for selected systems, pH4 Table 23. DNA titration example for SMCA experiments Table 24. SMCAdata for selected systems, pH 7 Table 25. Thermal Stability and Antigenbinding data for selected SMCA designs Table 26. SMCA data for selectedWT (vis-a-vis light chain tags) Table 27. Successful designs in bothLCCA (D3H44) and at least one tested SMCA system Table 30. Designlibrary Table 31. Core designs

1.-61. (canceled)
 62. An isolated antigen binding polypeptide constructcomprising at least a first heterodimer and a second heterodimer, thefirst heterodimer comprising a first immunoglobulin G (IgG) heavy chainpolypeptide sequence (H1) and a first immunoglobulin light chainpolypeptide sequence (L1), and binding to a first epitope; and thesecond heterodimer comprising a second immunoglobulin G (IgG) heavychain polypeptide sequence (H2) and a second immunoglobulin light chainpolypeptide sequence (L2), and binding to a second epitope, wherein atleast one of the H1 or L1 sequences of the first heterodimer is distinctfrom the corresponding H2 or L2 sequence of the second heterodimer, H1and H2 each comprise a heavy chain variable domain (V_(H) domain) and aheavy chain constant domain (CH1 domain), and L1 and L2 each comprise alight chain variable domain (V_(L) domain) and a light chain constantdomain (C_(L) domain); wherein H1 preferentially pairs with L1 ascompared to L2 and H2 preferentially pairs with L2 as compared to L1;wherein: a) H1 comprises amino acid substitution 139W; L1 comprisesamino acid substitution 116A, and L2 comprises amino acid substitution135W; b) H1 comprises amino acid substitution 139W; L1 comprises aminoacid substitutions 116A and 135A; H2 comprises amino acid substitutions139G and 145L and 179E and 190A, and L2 comprises amino acidsubstitutions 131R and 135W; c) H1 comprises amino acid substitution139W; L1 comprises amino acid substitutions 116S and 135A; H2 comprisesamino acid substitutions 139G and 145L and 179E and 190A, and L2comprises amino acid substitutions 131R and 135W; d) H1 comprises aminoacid substitution 139W; L1 comprises amino acid substitutions 116A and135A; H2 comprises amino acid substitutions 139G and 190A, and L2comprises amino acid substitutions 135W and 137A; e) H1 comprises aminoacid substitutions 37W and 139W; L1 comprises amino acid substitutions98A and 116A and 135A; H2 comprises amino acid substitutions 37A and103H and 139G and 190A, and L2 comprises amino acid substitutions 44Wand 135W; f) H1 comprises amino acid substitutions 37W and 103H and139W; L1 comprises amino acid substitutions 98L and 116A and 135A; H2comprises amino acid substitutions 139G and 190A, and L2 comprises aminoacid substitutions 98W and 135W; g) H1 comprises amino acidsubstitutions 39R and 139W; L1 comprises amino acid substitutions 38Eand 116A and 135V; H2 comprises amino acid substitutions 39E and 139Gand 190A, and L2 comprises amino acid substitutions 38R and 135W; h) H1comprises amino acid substitution 139W; L1 comprises amino acidsubstitutions 98W and 116A and 135A; H2 comprises amino acidsubstitutions 37W and 103H and 139G and 190A, and L2 comprises aminoacid substitutions 98L and 135W; i) H1 comprises amino acidsubstitutions 39R and 139W; L1 comprises amino acid substitutions 38Dand 116A and 135A; H2 comprises amino acid substitutions 39D and 139Gand 190A, and L2 comprises amino acid substitutions 38R and 135W; j) H1comprises amino acid substitutions 39D and 139W; L1 comprises amino acidsubstitution 38R and 116A and 135A; H2 comprises amino acidsubstitutions 39R and 139G and 190A, and L2 comprises amino acidsubstitutions 38D and 135W; k) H1 comprises amino acid substitutions139W and 145Y and 179E; L1 comprises amino acid substitution 116A and131K and 135A; H2 comprises amino acid substitutions 139G and 190A, andL2 comprises amino acid substitution 135W, or l) H1 comprises amino acidsubstitutions 139W and 186K; L1 comprises amino acid substitution 116Aand 124E and 135A and 180E; H2 comprises amino acid substitutions 139Gand 145T and 146G and 179E and 190A, and L2 comprises amino acidsubstitution 135W; wherein when both L1 and L2 are co-expressed with atleast one of H1 and H2, the pairing of H1-L1 to H1-L2 and the pairing ofH2-L2 to H2-L1 is greater than the pairing of H1-L1 to H1-L2 and thepairing of H2-L2 to H2-L1 in the absence of the amino acidsubstitutions, and wherein the numbering of amino acid residues isaccording to Kabat.
 63. The antigen binding polypeptide construct ofclaim 62, wherein H1 comprises amino acid substitution 139W; L1comprises amino acid substitution 116A, and L2 comprises amino acidsubstitution 135W.
 64. The antigen binding polypeptide construct ofclaim 62, wherein H1, H2, L1 and L2 are co-expressed in a cell or amammalian cell, or H1, H2, L1 and L2 are co-expressed in a cell-freeexpression system, or H1, H2, L1 and L2 are co-produced, or H1, H2, L1and L2 are co-produced via a redox production system.
 65. The antigenbinding polypeptide construct of claim 62, wherein the firstimmunoglobulin light chain polypeptide sequence and/or the secondimmunoglobulin light chain polypeptide sequence is a kappa light chainpolypeptide.
 66. The antigen binding polypeptide construct of claim 62,wherein the antigen binding polypeptide construct further comprises anFc comprising two heavy chain constant domain polypeptides, eachcomprising a CH3 sequence, wherein the heavy chain polypeptides arecoupled, with or without one or more linkers, to the first heterodimerand the second heterodimer.
 67. The antigen binding polypeptideconstruct of claim 66, wherein the Fc is a human Fc, a human IgG1 Fc, ahuman IgA Fc, a human IgG Fc, a human IgD Fc, a human IgE Fc, a humanIgM Fc, a human IgG2 Fc, a human IgG3 Fc, or a human IgG4 Fc.
 68. Theantigen binding polypeptide construct of claim 67, wherein the Fc is aheterodimeric Fc.
 69. The antigen binding polypeptide construct of claim68, wherein the Fc comprises one or more modifications in at least oneof the CH3 sequences that promote the formation of a heterodimeric Fcwith stability comparable to a wild-type homodimeric Fc.
 70. The antigenbinding polypeptide construct of claim 69, wherein: a) one of the CH3sequences comprises the amino acid substitutions L351Y, F405A, Y407V andthe other comprises the amino acid substitutions T366L, K392M, T394W; b)one of the CH3 sequences comprises the amino acid substitutions L351Y,F405A, Y407V and the other comprises the amino acid substitutions T366L,K392L, T394W; c) one of the CH3 sequences comprises the amino acidsubstitutions T350V, L351Y, F405A, Y407V and the other comprises theamino acid substitutions T350V, T366L, K392M, T394W; d) one of the CH3sequences comprises the amino acid substitutions T350V, L351Y, F405A,Y407V and the other comprises the amino acid substitutions T350V, T366L,K392L, T394W; or e) one of the CH3 sequences comprises the amino acidsubstitutions T350V, L351Y, S400E, F405A, Y407V and the other comprisesthe amino acid substitutions T350V, T366L, N390R, K392M, T394W, whereinthe numbering of amino acid residues of the CH3 sequences is accordingto the EU numbering system.
 71. The antigen binding polypeptideconstruct of claim 70, wherein the Fc further comprises at least one CH2sequence.
 72. The antigen binding polypeptide construct of claim 71,wherein the Fc comprises one or more modifications to promote selectivebinding of Fc-gamma receptors.
 73. The antigen binding polypeptideconstruct of claim 66, wherein the one or more linkers are one or morepolypeptide linkers, comprising one or more antibody hinge regions orone or more IgG1 hinge regions.
 74. The antigen binding polypeptideconstruct of claim 62, wherein the sequences of each of H1, H2, L1, andL2 are derived from human or humanized sequences.
 75. The antigenbinding polypeptide construct of claim 62, wherein the antigen bindingpolypeptide construct is multi-specific or bispecific.
 76. The antigenbinding polypeptide construct of claim 66, wherein the antigen bindingpolypeptide construct is conjugated to a therapeutic agent or drug. 77.A pharmaceutical composition comprising the antigen binding polypeptideconstruct of claim 62 and a pharmaceutically acceptable carrier.
 78. Anisolated polynucleotide or set of isolated polynucleotides comprising atleast one sequence that encodes the antigen binding polypeptideconstruct of claim
 62. 79. A vector or set of vectors comprising theisolated polynucleotide or set of isolated polynucleotides of claim 78.80. An isolated cell comprising the vector or set of vectors of claim79.
 81. A method of obtaining the antigen binding polypeptide constructof claim 62 from a host cell culture, the method comprising the stepsof: (a) obtaining a host cell culture comprising at least one host cellcomprising one or more nucleic acid sequences encoding the antigenbinding polypeptide construct; and (b) recovering the antigen bindingpolypeptide construct from the host cell culture.